System for Antibody Expression and Assembly

ABSTRACT

The present invention provides methods and compositions for expression and production of recombinant antibodies in a host cell system, such as prokaryotic and eukaryotic expression systems. Particularly contemplated are recombinant systems for temporally separated expression of light chain and heavy chain of antibodies. The antibody products including antibody fragments can be used in various aspects of biological research, diagnosis and medical treatment.

FIELD OF THE INVENTION

The present invention relates generally to the fields of molecularbiology and protein technology. More specifically, the inventionconcerns recombinantly produced antibodies and uses thereof.

BACKGROUND OF THE INVENTION

Recent years have seen increasing promises of using antibodies asdiagnostic and therapeutic agents for various disorders and diseases.Many research and clinical applications require large quantities offunctional antibodies or antibody fragments, thus calling for scaled-up,yet economic systems for antibody production. Particularly useful is therecombinant production of antibodies using a variety of expressionhosts, ranging from prokaryotes such as E. coli or B. subtilis, toyeast, plants, insect cells and mammalian cells. Kipriyanov and Little(1999) Mol. Biotech. 12:173-201.

Compared to other antibody production systems, bacteria, particularly E.coli, provides many unique advantages. The raw materials used (i.e.bacterial cells) are inexpensive and easy to grow, therefore reducingthe cost of products. Prokaryotic hosts grow much faster than, e.g.,mammalian cells, allowing quicker analysis of genetic manipulations.Shorter generation time and ease of scaling up also make bacterialfermentation a more attractive means for large quantity proteinproduction. The genomic structure and biological activity of manybacterial species including E. coli have been well-studied and a widerange of suitable vectors are available, making expression of adesirable antibody more convenient. Compared with eukaryotes, fewersteps are involved in the production process, including the manipulationof recombinant genes, stable transformation of multiple copies into thehost, expression induction and characterization of the products.Pluckthun and Pack (1997) Immunotech 3:83-105. In addition, E. colipermits a unique access to random approaches. Because of theunparalleled efficiency for transformation by plasmids or transfectionby phages, E. coli systems can be used for phage library construction ofmany types of antibody variants, which is particularly important infunctional genomic studies.

Various approaches have been used to make recombinant antibodies inbacteria. Like other heterologous proteins, antibody molecules can beobtained from bacteria either through refolding of inclusion bodiesexpressed in the cytoplasm, or through expression followed by secretionto the bacterial periplasm. The choice between secretion and refoldingis generally guided by several considerations. Secretion is usually thefaster and more commonly used strategy for producing antibodies.Kipriyanov and Little (1999), supra.

Opper et al., U.S. Pat. No. 6,008,023, describes an E. coli cytoplasmicexpression system, wherein antibody fragments (e.g., Fabs) are fusedwith an enzyme for use in targeted tumor therapy. Zemel-Dreasen et al.(1984) Gene 27:315-322 reports the secretion and processing of anantibody light chain in E. coli. Lo et al's PCT publication, WO93/07896, reports the E. coli production of a tetrameric antibodylacking the CH2 region in its heavy chain. The genes encoding the lightchain and the CH2-deleted heavy chain were constructed into the sameexpression vector, under the control of one single promoter.

Antibody expression in prokaryotic systems can be carried out indifferent scales. The shake-flask cultures (in the 2-5 liter-range)typically generate less than 5 mg/liter products. Carter et al. (1992)Bio/Technology 10:12-16 developed a high cell-density fermentationsystem in which high-level expression (up to 2 g/liter) of antibodyfragments was obtained. The gram per liter titers of Fab′ obtained byCarter et al. is due largely to higher cell densities resulting from themore precisely controlled environment of a fermentor than that of asimple shake flask. The system contains a dicistronic operon designed toco-express the light chain and heavy chain fragments. The dicistronicoperon is under the control of a single E. coli phoA promoter which isinducible by phosphate starvation. Each antibody chain is preceded bythe E. coli heat-stable enterotoxin II (stII) signal sequence to directsecretion to the periplasmic space. The system described by Carter etal. (1992) is further discussed herein below.

For general reviews of antibody production in E. coli, see Pluckthun andPack (1997) Immunotech 3:83-105; Pluckthun et al. (1996) in ANTIBODYENGINEERING: A PRACTICAL APPROACH, pp 203-252 (Oxford Press); Pluckthun(1994) in HANDBOOK OF EXP PHARMCOL VOL 3: THE PHARMCOL OF MONOCLONALANTIBODIES, pp 269-315 (ed. M. Rosenberg and G. P. Moore;Springer-Verlag, Berlin).

Many biological assays (such as X-ray crystallography) and clinicalapplications (such as protein therapy) require large amounts ofantibody. Accordingly, a need exists for high yield yet simple systemsfor producing properly assembled, soluble and functional antibodies.

SUMMARY OF THE INVENTION

The present invention provides novel methods and compositions forrecombinantly producing functional antibodies or antibody fragments inhost cells, such as prokaryotic or eukaryotic host cells. In oneembodiment, the invention provides a process for temporally separatingthe expression of light chain and heavy chain of an antibody in a hostcell such as a prokaryotic cell, thereby increasing the yield ofassembled, functional antibody molecules. In particular, the methodcomprises transforming the host cell with two separate translationalunits respectively encoding the light and heavy chains; culturing thecell under suitable conditions such that the light chain and heavy chainare expressed in a sequential fashion, thereby temporally separating theproduction of the light and heavy chains; and allowing the light andheavy chains to assemble into the functional antibody or fragmentthereof. In one preferred aspect, the temporally separated expression oflight and heavy chains is realized by utilizing two different promotersseparately controlling the light and heavy chains, wherein the differentpromoters are activated under different conditions. For example, DNAsencoding the light and heavy chains can be incorporated into a singleplasmid vector but are separated into two translational units, each ofwhich is controlled by a different promoter. One promoter (for example,a first promoter) can be either constitutive or inducible, whereas theother promoter (for example, a second promoter) is inducible. As such,when the host cells transformed with such vector are cultured underconditions suitable for activating one promoter (for example, the firstpromoter), only one chain (e.g., the light chain) is expressed. Then,after a desirable period of expression of the first chain (e.g. thelight chain), culturing conditions are changed to those suitable for theactivation of the other promoter (for example, the second promoter), andhence inducing the expression of the second chain (e.g. the heavychain). In one preferred embodiment, the light chain is expressed firstfollowed by the heavy chain. In another embodiment, the heavy chain isexpressed first followed by the light chain.

The invention also provides a recombinant vector for making an assembledfunctional antibody or fragment thereof in a prokaryotic or eukaryotichost cell, said vector comprising a first promoter preceding a firsttranslational unit encoding a secretion signal operably linked to alight chain; and a second promoter preceding a second translational unitencoding a secretion signal operably linked to a heavy chain. The firstand second promoters are inducible under different conditions.

Many prokaryotic and eukaryotic species can be used as hosts forantibody expression according to the invention. Preferably, aprokaryotic host is a gram-negative bacteria. More preferably, the hostis E. coli. In one aspect, the host cell is a genetically altered E.coli strain suitable for large quantity production of heterologousproteins. For example, the host cells may be an E. coli straincontaining mutant alleles for proteases, and or extra copies of the dsbgenes. Many known promoters, constitutive or inducible, are suitable foruse in the present invention, so long as they can be used effectively incombination with another promoter.

The methods and compositions of the invention can be used for largequantity production of a wide range of assembled antibody moleculesincluding intact antibody or antibody fragments such as Fab, Fab′,F(ab′)₂, F(ab′)₂-leucine zipper fusion, Fv and dsFv. Moreover, antibodymolecules of the invention can be of human, chimeric, humanized oraffinity-matured. The antibody can be specific to any appropriateantigen, preferably those biologically important polypeptides. Anantibody fragment may be fused to a dimerization domain, such as aleucine zipper domain.

Also contemplated are various diagnostic and therapeutic uses of theantibodies made according to the methods described herein. In onetherapeutic application, the recombinantly made antibody or fragmentthereof is used in combination with another therapeutic agent in atreatment.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a schematic representation of the construction of the antiCD18F(ab′)₂(-leucine zipper) plasmids pS1130 (single promoter) andpxCD18-7T3 (dual-promoter).

FIG. 2 depicts the insert nucleic acid sequence of the dual-promoterconstruct pxCD18-7T3.

FIG. 3 depicts the amino acid sequences encoded by the two translationalunits within the construct pxCD18-7T3. N-terminal STII secretion signalsequences are underlined.

FIG. 4 compares the yields of assembled F(ab′)₂ using the singlepromoter system (pS1130/59A7) and the dual promoter system(pxCD18-7T3/59A7).

FIG. 5 depicts anti-CD18 expression profiles with the single promoterexpression system (pS1130).

FIG. 6 depicts anti-CD18 expression profiles with the dual-promoterexpression system (pxCD18-7T3).

FIG. 7 compares the total heavy chain yields and assembly efficienciesof the single promoter system (pS1130) and the dual promoter system(pxCD18-7T3). The assembly efficiencies represent the fraction of heavychain assembled into F(ab′)₂ during the first 10 hours of heavy chainsynthesis.

FIG. 8 is a schematic of the anti-Tissue Factor IgG1 plasmids paTF130(PhoA/PhoA promoters) and pxTF-7T3FL (PhoA/TacII-promoters).

FIG. 9 depicts the insert nucleic acid sequence of thePhoA/TacII-promoter construct pxTF-7T3FL.

FIG. 10 depicts the amino acid sequences encoded by the twotranslational units within the construct pxTF-7T3FI. N-terminal STIIsecretion signal sequences are underlined.

FIGS. 11A and 11B are results of western blots under reduced (11A) ornon-reduced (11B) conditions, comparing the anti-Tissue Factor IgG1expressions using the same promoter system (PhoA/PhoA) and thedual-promoter system (PhoA/TacII).

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The principal embodiments of the present intention are based on thesurprising discovery that yields of properly assembled, solubleantibodies in a host cell system, such as an E. coli fermentationsystem, can be dramatically increased by temporally separating theinduction of the light chain and heavy chain expression. Using a novelrecombinant system wherein one chain (e.g., the light chain) wasexpressed prior to the induction of the second chain (e.g. the heavychain) expression, about two-fold improvement in titer of assembledantibody has been achieved over a comparable system wherein the lightand heavy chains were simultaneously expressed.

Antibodies have traditionally been produced in host cells, such as E.coli, using dicistronic vectors, in which genes encoding for light chainand heavy chain are under the control of a single promoter. Underculturing conditions suitable for the activation of the promoter, bothlight chain and heavy chain genes are expressed simultaneously. Forexample, Carter et al. (1992) Bio/Technology 10:163-167 describes adicistronic operon for light and heavy chain fragments under the controlof a single E. coli phoA promoter, which is inducible by phosphatestarvation. Each antibody chain is preceded by the E. coli heat-stableenterotoxin II (stII) signal sequence to direct secretion to theperiplasmic space. When this vector was used to carry out certainantibody production, especially when the expression levels of both lightchain and heavy chain were high, significant amounts of the individualchain molecules became aggregated, unable to be assembled into solubleand functional antibodies. The problems of aggregation in dicistronicvectors (having a single promoter for both light chain and heavy chainexpression) are further illustrated in the Examples provided hereinbelow.

Without being limited to a particular theory, the problem of aggregationcould be due, at least partly, to the limited ability of individualchain to fold under the conditions described above. Individual chains ofan antibody may behave differently during the process of expression,secretion and assembly into functional antibodies. For example, onechain (e.g. the light chain) may remain predominantly soluble afterbeing secreted alone into the periplasmic space of the host cells, whilethe other chain (e.g. the heavy chain) would become largely aggregatedand insoluble after secretion, unless it exists as part of an assembledantibody complex. Thus, earlier expression of the more soluble chain mayfacilitate the folding of the less soluble chain and subsequent assemblyof the two chains. Additionally, the host cell may have limited capacityfor translocating expressed polypeptides via its secretion apparatus.The limit of secretion capacity may be exceeded in certain situations,for example, where multiple polypeptides are expressed simultaneously,or where a great amount of precursor proteins are expressed by a vectorwith strong translational strength, or a protein of large size isexpressed, or any combination thereof. As the result, less matureproteins are secreted and available for assembly into the antibodycomplex.

Regardless of the mechanisms, the present invention provides a novel(prokaryotic or eukaryotic) system for antibody production withsignificantly increased yields of assembled, soluble and functionalantibody molecules. Using the dual promoter expression vector of theinvention, one chain is synthesized first. After a certain amount of thefirst chain has been expressed, the expression of the second chain canbe induced under a second promoter that is responsive to a changedculturing condition. The newly expressed second chain would then be ableto complex with the previously made first chain available to formsoluble antibodies or fragments thereof. Thus, by manipulating thetiming of the expression of the two-chains, more expressed antibodychains could be directed into the soluble antibody complex, increasingthe total yield thereof.

While the temporally separate expression system of the present inventionis mainly illustrated by the production of antibodies and fragmentsthereof, it should be understood that the approach described herein isapplicable in any system in which multiple protein units/chains are tobe produced and an intermediate or final protein complex requires properassembly of individual units/chains in order to be functional. Theapproach is especially useful for the production of protein complexescontaining immunoglobulin-like domains, such as antibodies, T-cellreceptors, class I and class II MHC molecules, integrins, CD8 and CD28molecules, and related fragments, derivatives, variants and fusionproteins thereof.

Antibody

The term “antibody” is used in the broadest sense and includesmonoclonal antibodies, polyclonal antibodies, multivalent antibodies,multispecific antibodies (e.g. bispecific antibodies), and antibodyfragments so long as they exhibit the desired biological activity. Anaturally occurring antibody comprises four polypeptide chains, twoidentical heavy (H) chains and two identical light (L) chainsinter-connected by disulfide bonds. Each heavy chain is comprised of aheavy chain variable region (V_(H)) and a heavy chain constant region,which in its native form is comprised of three domains, CH1, CH2 andCH3. Each light chain is comprised of a light chain variable region(V_(L)) and a light chain constant region. The light chain constantregion is comprised of one domain, CL. The V_(H) and V_(L) regions canbe further subdivided into regions of hypervariability, termedcomplementarity determining regions (CDR), interspersed with regionsthat are more conserved, termed framework regions (FR). Each V_(H) andV_(L) is composed of three CDRs and four FRs, arranged fromamino-terminus to carboxy-terminus in the following order: FR1, CDR1,FR2, CDR2, FR3, CDR3, FR4.

The light chains of antibodies from any vertebrate species can beassigned to one of two clearly distinct types, called kappa (κ) andlambda (λ), based on the amino acid sequences of their constant domains.

Depending on the amino acid sequences of the constant domains of theirheavy chains, antibodies (immunoglobulins) can be assigned to differentclasses. There are five major classes of immunoglobulins: IgA, IgD, IgE,IgG and IgM, and several of these may be further divided into subclasses(isotypes), e.g., IgG-1, IgG-2, IgA-1, IgA-2, and etc. The heavy chainconstant domains that correspond to the different classes ofimmunoglobulins are called α, δ, ε, γ, and μ, respectively. The subunitstructures and three-dimensional configurations of different classes ofimmunoglobulins are well known and described generally in, for example,Abbas et al. Cellular and Mol. Immunology, 4th ed. (2000).

An antibody may be part of a larger fusion molecule, formed by covalentor noncovalent association of the antibody or antibody portion with oneor more other proteins or peptides. Examples of such fusion proteinsinclude use of the streptavidin core region to make a tetrameric scFvmolecule (Kipriyanov et al. (1995) Human Antibodies and Hybridomas6:93-101) and use of a cysteine residue, a marker peptide and aC-terminal polyhistidine tag to make bivalent and biotinylated scFvmolecules (Kipriyanov, S. M., et al. (1994) Mol. Immunol. 31:1047-1058).

The present invention encompasses monoclonal antibodies. The term“monoclonal antibody” as used herein refers to an antibody obtained froma population of substantially homogeneous antibodies, i.e., theindividual antibodies comprising the population are identical except forpossible naturally occurring mutations that may be present in minoramounts. Monoclonal antibodies are highly specific, being directedagainst a single antigen. Furthermore, in contrast to polyclonalantibody preparations that typically include different antibodiesdirected against different determinants (epitopes), each monoclonalantibody is directed against a single determinant on the antigen.

The monoclonal antibodies herein specifically include “chimeric”antibodies in which a portion of the heavy and/or light chain isidentical with or homologous to corresponding sequences in antibodiesderived from a particular species or belonging to a particular antibodyclass or subclass, while the remainder of the chain(s) is identical withor homologous to corresponding sequences in antibodies derived fromanother species or belonging to another antibody class or subclass, aswell as fragments of such antibodies, so long as they exhibit thedesired biological activity (U.S. Pat. No. 4,816,567; and Morrison etal., Proc. Natl. Acad. Sci. USA 81:6851-6855 (1984)).

A “functional” or “biologically active” antibody is one capable ofexerting one or more of its natural activities in structural,regulatory, biochemical or biophysical events. For example, a functionalantibody may have the ability to specifically bind an antigen and thebinding may in turn elicit or alter a cellular or molecular event suchas signaling transduction or enzymatic activity. A functional antibodymay also block ligand activation of a receptor or act as an agonistantibody. The capability of an antibody to exert one or more of itsnatural activities depends on several factors, including proper foldingand assembly of the polypeptide chains. As used herein, the functionalantibodies generated by the disclosed methods are typicallyheterotetramers having two identical L chains and two identical H chainsthat are linked by multiple disulfide bonds and properly folded. In someaspects, the present invention encompasses blocking antibodies, antibodyantagonists and/or antibody agonists. A “blocking” antibody or anantibody “antagonist” is one which inhibits or reduces biologicalactivity of the antigen it binds. Such blocking can occur by any means,e.g. by interfering with: ligand binding to the receptor, receptorcomplex formation, tyrosine kinase activity of a tyrosine kinasereceptor in a receptor complex and/or phosphorylation of tyrosine kinaseresidue(s) in or by the receptor. For example, a VEGF antagonistantibody binds VEGF and inhibits the ability of VEGF to induce vascularendothelial cell proliferation. Preferred blocking antibodies orantagonist antibodies completely inhibit the biological activity of theantigen.

An “antibody agonist” is an antibody which binds and activates antigensuch as a receptor. Generally, the receptor activation capability of theagonist antibody will be at least qualitatively similar (and may beessentially quantitatively similar) to a native agonist ligand of thereceptor.

Antigen Specificity

The present invention is applicable to antibodies or antibody fragmentsof any appropriate antigen binding specificity. Preferably, theantibodies of the invention are specific to antigens that arebiologically important polypeptides. More preferably, the antibodies ofthe invention are useful for therapy or diagnosis of diseases ordisorders in a mammal. The antibodies or antibody fragments obtainedaccording to the present invention are particularly useful astherapeutic agents such as blocking antibodies, antibody agonists orantibody conjugates. Nonlimiting examples of therapeutic antibodiesinclude anti-VEGF, anti-IgE, anti-CD11, anti-CD18, anti-tissue factor,and anti-TrkC antibodies. Antibodies directed against non-polypeptideantigens (such as tumor-associated glycolipid antigens) are alsocontemplated.

The term “antigen” is well understood in the art and includes substanceswhich are immunogenic, i.e., immunogens, as well as substances whichinduce immunological unresponsiveness, or anergy, i.e., anergens. Wherethe antigen is a polypeptide, it may be a transmembrane molecule (e.g.receptor) or ligand such as a growth factor. Exemplary antigens includemolecules such as renin; a growth hormone, including human growthhormone and bovine growth hormone; growth hormone releasing factor;parathyroid hormone; thyroid stimulating hormone; lipoproteins;alpha-1-antitrypsin; insulin A-chain; insulin B-chain; proinsulin;follicle stimulating hormone; calcitonin; luteinizing hormone; glucagon;clotting factors such as factor VIIIC, factor IX, tissue factor (TF),and von Willebrands factor; anti-clotting factors such as Protein C;atrial natriuretic factor; lung surfactant; a plasminogen activator,such as urokinase or human urine or tissue-type plasminogen activator(t-PA); bombesin; thrombin; hemopoietic growth factor; tumor necrosisfactor-alpha and -beta; enkephalinase; RANTES (regulated on activationnormally T-cell expressed and secreted); human macrophage inflammatoryprotein (MIP-1-alpha); a serum albumin such as human serum albumin;Muellerian-inhibiting substance; relaxin A-chain; relaxin B-chain;prorelaxin; mouse gonadotropin-associated peptide; a microbial protein,such as beta-lactamase; DNase; IgE; a cytotoxic T-lymphocyte associatedantigen (CTLA), such as CTLA-4; inhibin; activin; vascular endothelialgrowth factor (VEGF); receptors for hormones or growth factors; proteinA or D; rheumatoid factors; a neurotrophic factor such as bone-derivedneurotrophic factor (BDNF), neurotrophin-3, -4, -5, or -6 (NT-3, NT-4,NT-5, or NT-6), or a nerve growth factor such as NGF-β; platelet-derivedgrowth factor (PDGF); fibroblast growth factor such as aFGF and bFGF;epidermal growth factor (EGF); transforming growth factor (TGF) such asTGF-alpha and TGF-beta, including TGF-β1, TGF-β2, TGF-β3, TGF-β4, orTGF-β5; insulin-like growth factor-I and -II (IGF-I and IGF-II);des(1-3)—IGF-I (brain IGF-I), insulin-like growth factor bindingproteins; CD proteins such as CD3, CD4, CD8, CD19 and CD₂₀;erythropoietin; osteoinductive factors; immunotoxins; a bonemorphogenetic protein (BMP); an interferon such as interferon-alpha,-beta, and -gamma; colony stimulating factors (CSFs), e.g., M-CSF,GM-CSF, and G-CSF; interleukins (ILs), e.g., IL-1 to IL-10; superoxidedismutase; T-cell receptors; surface membrane proteins; decayaccelerating factor; viral antigen such as, for example, a portion ofthe AIDS envelope; transport proteins; homing receptors; addressins;regulatory proteins; integrins such as CD11a, CD11b, CD11c, CD18, anICAM, VLA-4 and VCAM; a tumor associated antigen such as HER2, HER3 orHER4 receptor; and fragments of any of the above-listed polypeptides.

Preferred antigens for antibodies encompassed by the present inventioninclude CD proteins such as CD3, CD4, CD8. CD 11a, CD11b, CD18, CD19,CD₂O, CD34 and CD46; members of the ErbB receptor family such as the EGFreceptor, HER2, HER3 or HER4 receptor; cell adhesion molecules such asLFA-1, Mac1, p150.95, VLA-4, ICAM-1, VCAM, α4/β7 integrin, and αv/β3integrin including either α or β subunits thereof; growth factors suchas VEGF, tissue factor (TF), and TGF-β alpha interferon (α-IFN); aninterleukin, such as IL-8; IgE; blood group antigens Apo2, deathreceptor; flk2/flt3 receptor; obesity (OB) receptor; mpl receptor;CTLA-4; protein C etc. The most preferred targets herein are VEGF, TF,CD19, CD₂O, CD40, TGF-β, CD11a, CD18, Apo2 and C24.

Soluble antigens or fragments thereof, optionally conjugated to othermolecules, can be used as immunogens for generating antibodies. Fortransmembrane molecules, such as receptors, fragments of these molecules(e.g. the extracellular domain of a receptor) can be used as theimmunogen. Alternatively, cells expressing the transmembrane moleculecan be used as the immunogen. Such cells can be derived from a naturalsource (e.g. cancer cell lines) or may be cells which have beentransformed by recombinant techniques to express the transmembranemolecule. Other antigens and forms thereof useful for preparingantibodies will be apparent to those in the art.

The antibodies of the present invention may be monospecific, bispecific,trispecific or of greater multispecificity. Multispecific antibodies maybe specific to different epitopes of a single molecule or may bespecific to epitopes on different molecules. Methods for designing andmaking multispecific antibodies are known in the art. See, e.g.,Millstein et al. (1983) Nature 305:537-539; Kostelny et al. (1992) J.Immunol. 148:1547-1553; WO 93/17715.

Antibody Fragments

The present invention contemplates the prokaryotic or eukaryoticproduction of antibodies or antibody fragments. Many forms of antibodyfragments are known in the art and encompassed herein. “Antibodyfragments” comprise only a portion of an intact antibody, generallyincluding an antigen binding site of the intact antibody and thusretaining the ability to bind antigen. Examples of antibody fragmentsencompassed by the present definition include: (i) the Fab fragment,having VL, CL, VH and CH1 domains; (ii) the Fab′ fragment, which is aFab fragment having one or more cysteine residues at the C-terminus ofthe CH1 domain; (iii) the Fd fragment having V_(H) and CH1 domains; (iv)the Fd′ fragment having VH and CH1 domains and one or more cysteineresidues at the C-terminus of the CH1 domain; (v) the Fv fragment havingthe VL and VH domains of a single arm of an antibody; (vi) the dAbfragment (Ward et al., Nature 341, 544-546 (1989)) which consists of aVH domain; (vii) isolated CDR regions; (viii) F(ab′)₂ fragments, abivalent fragment including two Fab′ fragments linked by a disulphidebridge at the hinge region; (ix) single chain antibody molecules (e.g.single chain Fv; scFv) (Bird et al., Science 242:423-426 (1988); andHuston et al., PNAS (USA) 85:5879-5883 (1988)); (x) “diabodies” with twoantigen binding sites, comprising a heavy chain variable domain (VH)connected to a light chain variable domain (VL) in the same polypeptidechain (see, e.g., EP 404,097; WO 93/11161; and Hollinger et al., Proc.Natl. Acad. Sci. USA, 90:6444-6448 (1993)); (xi) “linear antibodies”comprising a pair of tandem Fd segments (VH-CH1-VH-CH1) which, togetherwith complementary light chain polypeptides, form a pair of antigenbinding regions (Zapata et al. Protein Eng. 8(10):1057-1062 (1995); andU.S. Pat. No. 5,641,870).

Moreover, the present invention contemplates antibody fragments that aremodified to improve their stability and or to create antibody complexeswith multivalency. For many medical applications, antibody fragmentsmust be sufficiently stable against denaturation or proteolysisconditions, and the antibody fragments should ideally bind the targetantigens with high affinity. A variety of techniques and materials havebeen developed to provide stabilized and or multivalent antibodyfragments. An antibody fragment of the invention may be fused to adimerization domain. In a preferred embodiment, the antibody fragmentsof the present invention are dimerized by the attachment of adimerization domain, such as leucine zippers.

“Leucine zipper” is a protein dimerization motif found in manyeukaryotic transcription factors where it serves to bring twoDNA-binding domains into appropriate juxtaposition for binding totranscriptional enhancer sequences. Dimerization of leucine zippersoccurs via the formation of a short parallel coiled coil, with a pair ofα-helices wrapped around each other in a superhelical twist. Zhu et al.(2000) J. Mol. Biol. 300:1377-1387. These coiled-coil structures, termed“leucine zippers” because of their preference for leucine in every 7thposition, have also been used as dimerization devices in other proteinsincluding antibodies. Hu et al. (1990) Science 250:1400-1403; Blondeland Bedouelle (1991) Protein Eng. 4:457. Several species of leucinezippers have been identified as particularly useful for dimeric andtetrameric antibody constructs. Pluckthun and Pack (1997) Immunotech.3:83-105; Kostelny et al. (1992) J. Immunol. 148:1547-1553.

Antibody Variants

Amino acid sequence modification(s) of antibodies or fragments thereofare contemplated. For example, it may be desirable to improve thebinding affinity and/or other biological properties of the antibody.Amino acid sequence variants of the antibody are prepared by introducingappropriate nucleotide changes into the antibody nucleic acid, or bypeptide synthesis. Such modifications include, for example, deletionsfrom, and/or insertions into and/or substitutions of, residues withinthe amino acid sequences of the antibody. Any combination of deletion,insertion, and substitution is made to arrive at the final construct,provided that the final construct possesses the desired characteristics.The amino acid alterations may be introduced in the subject antibodyamino acid sequence at the time that sequence is made.

A useful method for identification of certain residues or regions of theantibody that are preferred locations for mutagenesis is called “alaninescanning mutagenesis” as described by Cunningham and Wells (1989)Science, 244:1081-1085. Here, a residue or group of target residues areidentified (e.g., charged residues such as arg, asp, his, lys, and glu)and replaced by a neutral or negatively charged amino acid (mostpreferably alanine or polyalanine) to affect the interaction of theamino acids with antigen. Those amino acid locations demonstratingfunctional sensitivity to the substitutions then are refined byintroducing further or other variants at, or for, the sites ofsubstitution. Thus, while the site for introducing an amino acidsequence variation is predetermined, the nature of the mutation per seneed not be predetermined. For example, to analyze the performance of amutation at a given site, ala scanning or random mutagenesis isconducted at the target codon or region and the expressed antibodies arescreened for the desired activity.

Amino acid sequence insertions include amino- and/or carboxyl-terminalfusions ranging in length from one residue to polypeptides containing ahundred or more residues, as well as intrasequence insertions of singleor multiple amino acid residues. Examples of terminal insertions includean antibody with an N-terminal methionyl residue or the antibody fusedto a cytotoxic polypeptide. Other insertional variants of the antibodymolecule include the fusion to the N- or C-terminus of the antibody toan enzyme (e.g. for ADEPT) or a polypeptide which increases the serumhalf-life of the antibody.

Another type of variant is an amino acid substitution variant. Thesevariants have at least one amino acid residue in the antibody moleculereplaced by a different residue. The sites of greatest interest forsubstitutional mutagenesis include the hypervariable regions, but FRalterations are also contemplated. Conservative substitutions are shownin Table 1 under the heading of “preferred substitutions”. If suchsubstitutions result in a change in biological activity, then moresubstantial changes, denominated “exemplary substitutions” in Table 1,or as further described below in reference to amino acid classes, may beintroduced and the products screened. TABLE 1 Preferred Original ResidueExemplary Substitutions Substitutions Ala (A) val; leu; ile val Arg (R)lys; gln; asn lys Asn (N) gln; his; asp, lys; arg gln Asp (D) glu; asnglu Cys (C) ser; ala ser Gln (Q) asn; glu asn Glu (E) asp; gln asp Gly(G) ala ala His (H) asn; gln; lys; arg arg Ile (I) leu; val; met; ala;phe; norleucine leu Leu (L) norleucine; ile; val; met; ala; phe ile Lys(K) arg; gln; asn arg Met (M) leu; phe; ile leu Phe (F) leu; val; ile;ala; tyr tyr Pro (P) ala ala Ser (S) thr; cys cys Thr (T) ser ser Trp(W) tyr; phe tyr Tyr (Y) trp; phe; thr; ser phe Val (V) ile; leu; met;phe; ala; norleucine leu

Substantial modifications in the biological properties of the antibodyare accomplished by selecting substitutions that differ significantly intheir effect on maintaining (a) the structure of the polypeptidebackbone in the area of the substitution, for example, as a sheet orhelical conformation, (b) the charge or hydrophobicity of the moleculeat the target site, or (c) the bulk of the side chain. Naturallyoccurring residues are divided into groups based on common side-chainproperties:

(1) hydrophobic: norleucine, met, ala, vat, leu, ile;

(2) neutral hydrophilic: cys, ser, thr;

(3) acidic: asp, glu;

(4) basic: asn, gin, his, lys, arg;

(5) residues that influence chain orientation: gly, pro; and

(6) aromatic: trp, tyr, phe.

Non-conservative substitutions will entail exchanging a member of one ofthese classes for another class.

Any cysteine residue not involved in maintaining the proper conformationof the antibody also may be substituted, generally with serine, toimprove the oxidative stability of the molecule and prevent aberrantcrosslinking. Conversely, cysteine bond(s) may be added to the antibodyto improve its stability.

A particularly preferred type of substitutional variant involvessubstituting one or more hypervariable region residues of a parentantibody (e.g. a humanized or human antibody). Generally, the resultingvariant(s) selected for further development will have improvedbiological properties relative to the parent antibody from which theyare generated. A convenient way for generating such substitutionalvariants involves affinity maturation using phage display. Briefly,several hypervariable region sites (e.g. 6-7 sites) are mutated togenerate all possible amino acid substitutions at each site. Theantibodies thus generated are displayed from filamentous phage particlesas fusions to the gene III product of M13 packaged within each particle.The phage-displayed variants are then screened for their biologicalactivity (e.g. binding affinity) as herein disclosed. In order toidentify candidate hypervariable region sites for modification, alaninescanning mutagenesis can be performed to identify hypervariable regionresidues contributing significantly to antigen binding. Alternatively,or additionally, it may be beneficial to analyze a crystal structure ofthe antigen-antibody complex to identify contact points between theantibody and antigen. Such contact residues and neighboring residues arecandidates for substitution according to the techniques elaboratedherein. Once such variants are generated, the panel of variants issubjected to screening as described herein and antibodies with superiorproperties in one or more relevant assays may be selected for furtherdevelopment.

Nucleic acid molecules encoding amino acid sequence variants of theantibody are prepared by a variety of methods known in the art. Thesemethods include, but are not limited to, isolation from a natural source(in the case of naturally occurring amino acid sequence variants) orpreparation by oligonucleotide-mediated (or site-directed) mutagenesis,PCR mutagenesis, and cassette mutagenesis of an earlier prepared variantor a non-variant version of the antibody.

It may be desirable to introduce one or more amino acid modifications inan Fc region of the antibody of the invention, thereby generating a Fcregion variant. The Fc region variant may comprise a human Fc regionsequence (e.g., a human IgG1, IgG2, IgG3 or IgG4 Fc region) comprisingan amino acid modification (e.g. a substitution) at one or more aminoacid positions.

In one embodiment, the Fc region variant may display altered neonatal Fcreceptor (FcRn) binding affinity. Such variant Fc regions may comprisean amino acid modification at any one or more of amino acid positions238, 252, 253, 254, 255, 256, 265, 272, 286, 288, 303, 305, 307, 309,311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 386, 388, 400,413, 415, 424, 433, 434, 435, 436, 439 or 447 of the Fc region, whereinthe numbering of the residues in the Fc region is that of the EU indexas in Kabat. Fc region variants with reduced binding to an FcRn maycomprise an amino acid modification at any one or more of amino acidpositions 252, 253, 254, 255, 288, 309, 386, 388, 400, 415, 433, 435,436, 439 or 447 of the Fc region, wherein the numbering of the residuesin the Fc region is that of the EU index as in Kabat. Theabove-mentioned Fc region variants may, alternatively, display increasedbinding to FcRn and comprise an amino acid modification at any one ormore of amino acid positions 238, 256, 265, 272, 286, 303, 305, 307,311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 413, 424 or 434of the Fc region, wherein the numbering of the residues in the Fc regionis that of the EU index as in Kabat.

The Fc region variant with reduced binding to an FcγR may comprise anamino acid modification at any one or more of amino acid positions 238,239, 248, 249, 252, 254, 265, 268, 269, 270, 272, 278, 289, 292, 293,294, 295, 296, 298, 301, 303, 322, 324, 327, 329, 333, 335, 338, 340,373, 376, 382, 388, 389, 414, 416, 419, 434, 435, 437, 438 or 439 of theFc region, wherein the numbering of the residues in the Fc region isthat of the EU index as in Kabat.

For example, the Fc region variant may display reduced binding to anFcγRI and comprise an amino acid modification at any one or more ofamino acid positions 238, 265, 269, 270, 327 or 329 of the Fc region,wherein the numbering of the residues in the Fc region is that of the EUindex as in Kabat.

The Fc region variant may display reduced binding to an FcγRII andcomprise an amino acid modification at any one or more of amino acidpositions 238, 265, 269, 270, 292, 294, 295, 298, 303, 324, 327, 329,333, 335, 338, 373, 376, 414, 416, 419, 435, 438 or 439 of the Fcregion, wherein the numbering of the residues in the Fc region is thatof the EU index as in Kabat.

The Fc region variant of interest may display reduced binding to anFcγRIII and comprise an amino acid modification at one or more of aminoacid positions 238, 239, 248, 249, 252, 254, 265, 268, 269, 270, 272,278, 289, 293, 294, 295, 296, 301, 303, 322, 327, 329, 338, 340, 373,376, 382, 388, 389, 416, 434, 435 or 437 of the Fc region, wherein thenumbering of the residues in the Fc region is that of the EU index as inKabat.

Fc region variants with altered (i.e. improved or diminished) C1qbinding and/or Complement Dependent Cytotoxicity (CDC) are described inWO99/51642. Such variants may comprise an amino acid substitution at oneor more of amino acid positions 270, 322, 326, 327, 329, 331, 333 or 334of the Fc region. See, also, Duncan & Winter Nature 322:738-40 (1988);U.S. Pat. No. 5,648,260; U.S. Pat. No. 5,624,821; and WO94/29351concerning Fc region variants.

Human, Humanized or Affinity Matured Antibodies

A “human antibody” is one which possesses an amino acid sequence whichcorresponds to that of an antibody produced by a human and/or has beenmade using any of the techniques for making human antibodies asdisclosed herein. This definition of a human antibody specificallyexcludes a humanized antibody comprising non-human antigen-bindingresidues.

The present invention encompasses both human and humanized antibodies.“Humanized” forms of non-human (e.g., murine) antibodies are chimericantibodies that contain minimal sequence derived from non-humanimmunoglobulin. For the most part, humanized antibodies are humanimmunoglobulins (recipient antibody) in which residues from ahypervariable region of the recipient are replaced by residues from ahypervariable region of a non-human species (donor antibody) such asmouse, rat, rabbit or nonhuman primate having the desired specificity,affinity, and capacity. In some instances, framework region (FR)residues of the human immunoglobulin are replaced by correspondingnon-human residues. Furthermore, humanized antibodies may compriseresidues that are not found in the recipient antibody or in the donorantibody. These modifications are made to further refine antibodyperformance. In general, the humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the hypervariable loops correspondto those of a non-human immunoglobulin and all or substantially all ofthe FRs are those of a human immunoglobulin sequence. The humanizedantibody optionally will also comprise at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin. For further details, see Jones et al., Nature321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); andPresta, Curr. Op. Struct. Biol. 2:593-596 (1992). See also the followingreview articles and references cited therein: Vaswani and Hamilton, Ann.Allergy, Asthma &Immunol. 1:105-115 (1998); Harris, Biochen. Soc.Transactions 23:1035-1038 (1995); Hurle and Gross, Curr. Op. Biotech.5:428-433 (1994).

An “affinity matured” antibody is one with one or more alterations inone or more CDRs thereof which result in an improvement in the affinityof the antibody for antigen, compared to a parent antibody which doesnot possess those alteration(s). Preferred affinity matured antibodieswill have nanomolar or even picomolar affinities for the target antigen.Affinity matured antibodies are produced by procedures known in the art.Marks et al. Bio/Technology 10:779-783 (1992) describes affinitymaturation by VH and VL domain shuffling. Random mutagenesis of CDRand/or framework residues is described by: Barbas et al. Proc Nat. Acad.Sci, USA 91:3809-3813 (1994); Schier et al. Gene 169:147-155 (1995);Yelton et al. J. Immunol. 155:1994-2004 (1995); Jackson et al., J.Immunol. 154(7):3310-9 (1995); and Hawkins et al., J. Mol. Biol.226:889-896 (1992).

Various methods for humanizing non-human antibodies are known in theart. For example, humanization can be essentially performed followingthe method of Winter and co-workers (Jones et al. (1986) Nature321:522-525; Riechmann et al. (1988) Nature 332:323-327; Verhoeyen etal. (1988) Science 239:1534-1536), by substituting hypervariable regionsequences for the corresponding sequences of a human antibody.Accordingly, such “humanized” antibodies are chimeric antibodies (U.S.Pat. No. 4,816,567) wherein substantially less than an intact humanvariable domain has been substituted by the corresponding sequence froma non-human species. In practice, humanized antibodies are typicallyhuman antibodies in which some hypervariable region residues andpossibly some FR residues are substituted by residues from analogoussites in rodent antibodies.

The choice of human variable domains, both light and heavy, to be usedin making the humanized antibodies is very important to reduceantigenicity. According to the so-called “best-fit” method, the sequenceof the variable domain of a rodent antibody is screened against theentire library of known human variable-domain sequences. The humansequence which is closest to that of the rodent is then accepted as thehuman framework for the humanized antibody (Sims et al. (1993) J.Immunol. 151:2296; Chothia et al. (1987) J. Mol. Biol. 196:901. Anothermethod uses a particular framework derived from the consensus sequenceof all human antibodies of a particular subgroup of light or heavychains. The same framework may be used for several different humanizedantibodies (Carter et al. (1992) Proc. Natl. Acad. Sci. USA, 89:4285;Presta et al. (1993) J Immunol., 151:2623.

It is further important that antibodies be humanized with retention ofhigh affinity for the antigen and other favorable biological properties.To achieve this goal, according to a preferred method, humanizedantibodies are prepared by a process of analysis of the parentalsequences and various conceptual humanized products usingthree-dimensional models of the parental and humanized sequences.Three-dimensional immunoglobulin models are commonly available and arefamiliar to those skilled in the art. Computer programs are availablewhich illustrate and display probable three-dimensional conformationalstructures of selected candidate immunoglobulin sequences. Inspection ofthese displays permits analysis of the likely role of the residues inthe functioning of the candidate immunoglobulin sequence, i.e., theanalysis of residues that influence the ability of the candidateimmunoglobulin to bind its antigen. In this way, FR residues can beselected and combined from the recipient and import sequences so thatthe desired antibody characteristic, such as increased affinity for thetarget antigen(s), is achieved. In general, the hypervariable regionresidues are directly and most substantially involved in influencingantigen binding.

Antibody Derivatives

The antibodies and antibody variants of the present invention can befurther modified to contain additional nonproteinaceous moieties thatare known in the art and readily available. Derivatizations areespecially useful for improving or restoring biological properties ofthe antibody or fragments thereof. For example, PEG modification ofantibody fragments can alter the stability, in vivo circulating halflife, binding affinity, solubility and resistance to proteolysis.

Preferably, the moieties suitable for derivatization of the antibody arewater soluble polymers. Non-limiting examples of water soluble polymersinclude, but are not limited to, polyethylene glycol (PEG), copolymersof ethylene glycol/propylene glycol, carboxymethylcellulose, dextran,polyvinyl alcohol, polyvinyl pyrrolidone, poly-1, 3-dioxolane,poly-1,3,6-trioxane, ethylene/maleic anhydride copolymer, polyaminoacids(either homopolymers or random copolymers), and dextran or poly(n-vinylpyrrolidone)polyethylene glycol, propropylene glycol homopolymers,prolypropylene oxide/ethylene oxide co-polymers, polyoxyethylatedpolyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof.Polyethylene glycol propionaldehyde may have advantages in manufacturingdue to its stability in water. The polymer may be of any molecularweight, and may be branched or unbranched. The number of polymersattached to the antibody may vary, and if more than one polymer isattached, they can be the same or different molecules. In general, thenumber and or type of polymers used for derivatization can be determinedbased on considerations including, but not limited to, the particularproperties or functions of the antibody to be improved, whether theantibody derivative will be used in a therapy under defined conditions.

In general, the antibody or antibody fragment produced by a prokaryoticexpression system as described herein is aglycosylated and lacksdetectable effector activities of the Fc region. In some instances, itmay be desirable to at least partially restore one or more effectorfunctions of the native antibody. Accordingly, the present inventioncontemplates a method for restoring the effector function(s) byattaching suitable moieties to identified residue sites in the Fc regionof the aglycosylated antibody. A preferred moiety for this purpose isPEG, although other carbohydrate polymers can also be used. Pegylationmay be carried out by any of the pegylation reactions known in the art.See, for example, EP 0401384; EP 0154316; WO 98/48837. In oneembodiment, cysteine residues are first substituted for residues atidentified positions of the antibody, such as those positions whereinthe antibody or antibody variant is normally glycosylated or thosepositions on the surface of the antibody. Preferably, the cysteine issubstituted for residue(s) at one or more positions 297, 298, 299, 264,265 and 239 (numbering according to the EU index as in Kabat). Afterexpression, the cysteine substituted antibody variant can have variousforms of PEG (or pre-synthesized carbohydrate) chemically linked to thefree cysteine residues.

Antibody Production

Vector Construction

The term “vector,” as used herein, is intended to refer to a nucleicacid molecule capable of transporting another nucleic acid to which ithas been linked. One type of vector is a “plasmid”, which refers to acircular double stranded DNA loop into which additional DNA segments maybe ligated. Another type of vector is a viral vector, wherein additionalDNA segments may be ligated into the viral genome. Certain vectors arecapable of autonomous replication in a host cell into which they areintroduced (e.g., bacterial vectors having a bacterial origin ofreplication and episomal mammalian vectors). Other vectors (e.g.,non-episomal mammalian vectors) can be integrated into the genome of ahost cell upon introduction into the host cell, and thereby arereplicated along with the host genome. Moreover, certain vectors arecapable of directing the expression of genes to which they are operablylinked. Such vectors are referred to herein as “recombinant expressionvectors” (or simply, “recombinant vectors”). In general, expressionvectors of utility in recombinant DNA techniques are often in the formof plasmids. In the present specification, “plasmid” and “vector” may beused interchangeably as the plasmid is the most commonly used form ofvector.

The term “translational unit,” as used herein, is intended to refer to agenetic element comprising the nucleotide sequence coding for apolypeptide chain and adjacent control regions. “Adjacent controlregions” include, for example, a translational initiation region (TIR;as defined herein below) and a termination region.

The “translation initiation region” or TIR as used herein refers to anucleic acid region providing the efficiency of translational initiationof a gene of interest. In general, a TIR within a particulartranslational unit encompasses the ribosome binding site (RBS) andsequences 5′ and 3′ to RBS. The RBS is defined to contain, minimally,the Shine-Dalgarno region and the start codon (AUG). Accordingly, a TIRalso includes at least a portion of the nucleic acid sequence to betranslated. Preferably, a TIR includes the sequence encoding a secretionsignal peptide that precedes the sequence encoding for the light orheavy chain within a translational unit. A TIR variant contains sequencevariants (particularly substitutions) within the TIR region that alterthe efficiency of the TIR, such as its translational strength as definedherein below. Preferably, a TIR variant of the invention containssequence substitutions within the first 2 to about 14, preferably about4 to 12, more preferably about 6 codons of the signal sequence thatprecedes the sequence encoding for the light or heavy chain within atranslational unit.

The term “translational strength” as used herein refers to a measurementof a secreted polypeptide in a control system wherein one or morevariants of a TIR is used to direct secretion of a polypeptide and theresults compared to the wild-type TIR or some other control under thesame culture and assay conditions. Without being limited to any onetheory, “translational strength” as used herein can include, forexample, a measure of mRNA stability, efficiency of ribosome binding tothe ribosome binding site, and mode of translocation across a membrane.

“Secretion signal sequence” or “secretion signal peptide” refers to ashort amino acid sequence that can be used to direct a newly synthesizedprotein of interest through a cellular membrane, for example, the innermembrane or both inner and outer membranes of prokaryotes. As such, inprokaryotic cells, for example, the protein of interest such as thelight or heavy chain polypeptide is secreted into the periplasm of theprokaryotic host cells or into the culture medium. The secretion signalsequences may be endogenous to the host cells, or they may be exogenous,including signal sequences native to the polypeptide to be expressed.Secretion signal sequences are typically at the N-terminal portion of apolypeptide and are typically removed enzymatically between biosynthesisand secretion of the polypeptide from the cytoplasm. Thus, the secretionsignal sequence is usually not present in the final protein product.

The term “host cell” (or “recombinant host cell”), as used herein, isintended to refer to a cell that has been genetically altered, or iscapable of being genetically altered by introduction of an exogenouspolynucleotide, such as a recombinant plasmid or vector. It should beunderstood that such terms are intended to refer not only to theparticular subject cell but to the progeny of such a cell. Becausecertain modifications may occur in succeeding generations due to eithermutation or environmental influences, such progeny may not, in fact, beidentical to the parent cell, but are still included within the scope ofthe term “host cell” as used herein.

DNA sequences encoding the light and heavy chains of the antibodymolecule of the invention can be obtained using standard recombinant DNAtechniques. Desired DNA sequences may be isolated and sequenced fromantibody producing cells such as hybridoma cells. Alternatively, the DNAcan be synthesized using nucleotide synthesizer or PCR techniques. Onceobtained, DNAs encoding the light and heavy chains are inserted into arecombinant vector capable of replicating, expressing and secretingheterologous polynucleotides in prokaryotic or eukaryotic hosts. Manyvectors that are available and known in the art can be used for thepurpose of the present invention. Selection of an appropriate vectorwill depend mainly on the size of nucleic acids to be inserted and theparticular host cell to be transformed with the vector.

In general, recombinant vectors containing replicon and controlsequences which are derived from species compatible with the host cellare used as parent vectors for the construction of the specific vectorsof the present invention. The vector ordinarily carries as backbonecomponents an origin of replication site as well as marking sequenceswhich are capable of providing phenotypic selection in transformedcells. The origin of replication site is a nucleic acid sequence thatenables the vector to replicate in one or more selected host cells.Generally, in cloning vectors this sequence is one that enables thevector to replicate independently of the host chromosomal DNA, andincludes origins of replication or autonomously replicating sequences.Such sequences are well known for a variety of bacteria, yeast, andviruses. The origin of replication from the plasmid pBR322 is suitablefor most Gram-negative bacteria.

Expression and cloning vectors may contain a selection gene, also termeda selectable marker. Typical selection genes encode proteins that (a)confer resistance to antibiotics or other toxins, e.g., ampicillin,neomycin, methotrexate, or tetracycline, (b) complement auxotrophicdeficiencies, or (c) supply critical nutrients not available fromcomplex media, e.g., the gene encoding D-alanine racemase for Bacilli.One example of a selection scheme utilizes a drug to arrest growth of ahost cell. Those cells that are successfully transformed with aheterologous gene produce a protein conferring drug resistance and thussurvive the selection regimen. An example of plasmid vector suitable forE. coli transformation is pBR322. pBR322 contains genes encodingampicillin (Amp) and tetracycline (Tet) resistance and thus provideseasy means for identifying transformed cells. Derivatives of pBR322 orother microbial plasmids or bacteriophage may also be used as parentvectors. Examples of pBR322 derivatives used for expression ofparticular antibodies are described in detail in Carter et al., U.S.Pat. No. 5,648,237.

In addition, phage vectors containing replicon and control sequencesthat are compatible with the host microorganism can be used astransforming vectors in connection with these hosts. For example,bacteriophage such as kGEM™-11 may be utilized in making a recombinantvector which can be used to transform susceptible host cells such as E.coli LE392.

According to one embodiment, the recombinant vector of the inventioncomprises at least two translational units, one for the light chainexpression and the other for the heavy chain expression. Moreover, thetwo translational units for light chain and heavy chain are under thecontrol of different promoters. Promoters are untranslated sequenceslocated upstream (5′) to the start of a coding sequence (generallywithin about 100 to 1000 bp) that control its expression. Such promoterstypically fall into two classes, inducible and constitutive. Induciblepromoters are promoters that initiate increased levels of transcriptionfrom DNA under their control in response to some change in cultureconditions, e.g. the presence or absence of a nutrient or a change intemperature or pH.

For the purpose of the present invention, either constitutive orinducible promoters can be used as the first promoter controlling thefirst chain expression in time, and inducible promoters are used as thesecond promoter controlling the subsequent second chain expression. In apreferred embodiment, both the first promoter and the second promoterare inducible promoters under tight regulation. A large number ofpromoters recognized by a variety of potential host cells are wellknown. The selected promoter sequence can be isolated from the sourceDNA via restriction enzyme digestion and inserted into the vector of theinvention. Alternatively the selected promoter sequences can besynthesized. Both the native promoter sequence and many heterologouspromoters may be used to direct amplification and/or expression of atarget gene. However, heterologous promoters are preferred, as theygenerally permit greater transcription and higher yields of expressedtarget gene as compared to the native target polypeptide promoter.

Promoters suitable for use with prokaryotic hosts include the PhoApromoter, the α-lactamase and lactose promoter systems, a tryptophan(trp) promoter system and hybrid promoters such as the tac or the trcpromoter. However, other promoters that are functional in bacteria (suchas other known bacterial or phage promoters) are suitable as well. Theirnucleotide sequences have been published, thereby enabling a skilledworker operably to ligate them to translational units encoding thetarget light and heavy chains using linkers or adaptors to supply anyrequired restriction sites (Siebenlist et al. (1980) Cell 20: 269). Morepreferred promoters for use in this invention are the PhoA promoter andthe TacII promoter. Promoters that are functional in eukaryotic hostcells are well known in the art, for example as described in U.S. Pat.No. 6,331,415. Examples of such promoters may include those derived frompolyoma, Adenovirus 2 or Simian Virus 40 (SV40).

Each translational unit of the recombinant vector of the inventioncontains additional untranslated sequences necessary for sufficientexpression of the inserted genes. Such essential sequences ofrecombinant vectors are known in the art and include, for example, theShine-Dalgarno region located 5′-to the start codon and transcriptionterminator (e.g., λto) located at the 3′-end of the translational unit.

Each translational unit of the recombinant vector further comprises asignal sequence component that directs secretion of the expressed chainpolypeptides across a membrane. In general, the secretion signalsequence may be a component of the vector, or it may be a part of thetarget polypeptide DNA that is inserted into the vector. The secretionsignal sequence selected for the purpose of this invention should be onethat is recognized and processed (i.e. cleaved by a signal peptidase) bythe host cell. For prokaryotic host cells that do not recognize andprocess the signal sequences native to the heterologous polypeptides,the signal sequence is substituted by a prokaryotic signal sequenceselected, for example, from the group consisting of the alkalinephosphatase, penicillinase, Ipp, or heat-stable enterotoxin II (STII)leaders, LamB, PhoE, PelB, OmpA and MBP. In a preferred embodiment ofthe invention, the signal sequences used in both translational units ofthe expression system are STII signal sequences or variants thereof.Preferably, the DNA encoding for such signal sequence is ligated inreading frame to the 5′-end of DNA encoding the light or heavy chain,resulting in a fusion polypeptide. Once secreted out of the cytoplasm ofthe host cell, the signal peptide sequence is enzymatically cleaved offfrom the mature polypeptide.

In some aspects of the invention, in addition to the timing of theexpression, the quantitative ratio of light and heavy chain expressionis also modulated in order to maximize the yield of secreted andcorrectly assembled antibodies or fragments thereof. Such modulation isaccomplished by simultaneously modulating translational strengths forlight and heavy chains on the recombinant vector of the invention. Onetechnique for modulating translational strength is disclosed in Simmonset al. U.S. Pat. No. 5,840,523. Briefly, the approach utilizes variantsof the translational initiation region (TIR) within a translationalunit. For a given TIR, a series of amino acid or nucleic acid sequencevariants can be created with a range of translational strengths, therebyproviding a convenient means by which to adjust this factor for thedesired expression level of the specific chain. TIR variants can begenerated by conventional mutagenesis techniques that result in codonchanges which can alter the amino acid sequence, although silent changesin the nucleotide sequence (as described below) are preferred.Alterations in the TIR can include, for example, alterations in thenumber or spacing of Shine-Dalgarno sequences, along with alterations inthe signal sequence. One preferred method for generating mutant signalsequences is the generation of a “codon bank” at the beginning of acoding sequence that does not change the amino acid sequence of thesignal sequence (i.e., the changes are silent). This can be accomplishedby changing the third nucleotide position of each codon; additionally,some amino acids, such as leucine, serine, and arginine, have multiplefirst and second positions that can add complexity in making the bank.This method of mutagenesis is described in detail in Yansura et al.(1992) METHODS: A Companion to Methods in Enzymol. 4:151-158.

Preferably, a set of vectors is generated with a range of TIR strengthsfor each translational unit therein. This limited set provides acomparison of expression levels of each chain as well as the yield ofantibody products under various TIR strength combinations. TIR strengthscan be determined by quantifying the expression level of a reporter geneas described in detail in Simmons et al. U.S. Pat. No. 5,840,523. Forthe purpose of this invention, the translational strength combinationfor a particular pair of TIRs within a vector is represented by(N-light, M-heavy), wherein N is the relative TIR strength of lightchain and M is the relative TIR strength of heavy chain. For example,(7-light, 3-heavy) means the vector provides a relative TIR strength ofabout 7 for light chain expression and a relative TIR strength of about3 for heavy chain expression. Based on the translational strengthcomparison, the desired individual TIRs are selected to be combined inthe expression vector constructs of the invention.

Construction of suitable vectors containing one or more of the abovelisted components employs standard ligation techniques and or othermolecular cloning techniques know in the art. Isolated plasmids or DNAfragments are cleaved, tailored, and re-ligated in the form desired togenerate the plasmids required.

For analysis to confirm correct sequences in plasmids constructed, theligation mixtures are used to transform an E. coli strain, andsuccessful transformants are selected by ampicillin or tetracyclineresistance where appropriate. Plasmids from the transformants areprepared, analyzed by restriction endonuclease digestion, and/orsequenced by the method of Sanger et al.(1977) Proc. Natl. Acad. Sci.USA 74:5463-5467 or Messing et al.(1981) Nucleic Acids Res. 9:309, or bythe method of Maxam et al. (1980) Methods in Enzymology 65:499. A numberof automated sequencers commercially available can be used to sequencethe plasmids. For example, the ABI PRISM 3700 DNA Analyzer (AppliedBiosystems, Foster City, Calif.) is an automated capillaryelectrophoresis sequencer that analyzes fluorescently labeled DNAfragments. Instructions for the preparation of samples are provided inthe Sequencing Chemistry Guide that accompanies the instrument.

Prokaryotic host cells suitable for expressing antibodies of theinvention include Archaebacteria and Eubacteria, such as Gram-negativeor Gram-positive organisms. Examples of useful bacteria includeEscherichia (e.g., E. coli), Bacilli (e.g., B. subtilis),Enterobacteria, Pseudomonas species (e.g., P. aeruginosa), Salmonellatyphimurium, Serratia marcescans, Klebsiella, Proteus, Shigella,Rhizobia, Vitreoscilla, or Paracoccus. Preferably, gram-negative cellsare used. More preferably, E. coli cells are used as hosts for theinvention.

Many E. coli strains are suitable as expression hosts herein or asparent hosts from which modified expression hosts can be created. E.coli strains that are known and available in the art include, but notlimited to, E. coli W3110 (ATCC 27,325), E. coli 294 (ATCC 31,446), E.coli B, E. coli 1776 (ATCC 31,537) and E. coli RV308(ATCC 31,608).Mutant cells of any of the above-mentioned bacteria may also beemployed. It is, of course, necessary to select the appropriate bacteriataking into consideration replicability of the replicon in the cells ofa bacterium. For example, E. coli, Serratia, or Salmonella species canbe suitably used as the host when well known plasmids such as pBR322,pBR325, pACYC177, or pKN410 are used to supply the replicon. Preferablythe host cell should secrete minimal amounts of proteolytic enzymes, andadditional protease inhibitors may desirably be incorporated in the cellculture.

Suitable eukaryotic host cells are also known in the art. For example,host cells may include yeast, VERO, HeLa, CH0, W138, BHK, COS-7 and MDCKcells.

Transformation and Growth of the Host Cells

Host cells are transformed or transfected (the terms “transformed” and“transfected” are used interchangeably herein) with the above-describedrecombinant vectors and cultured in conventional nutrient media modifiedas appropriate for inducing promoters, selecting transformants, oramplifying the genes encoding the desired sequences.

Transformation means introducing DNA into the prokaryotic host so thatthe DNA is replicable, either as an extrachromosomal element or bychromosomal integrant. Depending on the host cell used, transformationis done using standard techniques appropriate to such cells. The calciumtreatment employing calcium chloride is generally used for bacterialcells that contain substantial cell-wall barriers. Another method fortransformation employs polyethylene glycol/DMSO. Yet another techniqueused is electroporation. Transfection refers to the taking up of anexpression vector by a host cell whether or not any coding sequences arein fact expressed. Numerous methods of transfection are known to theordinarily skilled artisan, for example, Ca PO₄ precipitation andelectroporation. Successful transfection is generally recognized whenany indication of the operation of this vector occurs within the hostcell.

Prokaryotic cells used to produce the polypeptides of the invention aregrown in media known in the art and suitable for culture of the selectedhost cells. Examples of suitable media include luria broth (LB) plusnecessary nutrient supplements. In preferred embodiments, the media alsocontains a selection agent, chosen based on the construction of theexpression vector, to selectively permit growth of prokaryotic cellscontaining the expression vector. For example, ampicillin is added tomedia for growth of cells expressing ampicillin resistant gene. Anynecessary supplements besides carbon, nitrogen, and inorganic phosphatesources may also be included at appropriate concentrations introducedalone or as a mixture with another supplement or medium such as acomplex nitrogen source. Optionally the culture medium may contain oneor more reducing agents selected from the group consisting ofglutathione, cysteine, cystamine, thioglycollate, dithioerythritol anddithiothreitol.

The prokaryotic host cells are cultured at suitable temperatures. For E.coli growth, for example, the preferred temperature ranges from about20° C. to about 39° C., more preferably from about 25° C. to about 37°C. even more preferably at about 30° C. The pH of the medium may be anypH ranging from about 5 to about 9, depending mainly on the hostorganism. For E. coli, the pH is preferably from about 6.8 to about 7.4,and more preferably about 7.0.

Eukaryotic host cells used to produce antibodies of the invention can becultured in a variety of media known in the art. For example,commercially available media such as Ham's F10 (Sigma), MinimalEssential Medium ((MEM), Sigma), RPMI-1640 (Signma), and Dulbecco'sModified Eagle's Medium ((DMEM), Sigma) are suitable for culturingmammalian eukaryotic host cells. In addition, any of the media describedin Ham and Wallace,. Meth. Enz., 58: 44 (1979), Barnes and Sato, Anal.Biochem., 102:255 (1980), U.S. Pat. Nos. 4.767,704; 4,657.866;4.927,762; or 4,560,655; WO 90/03430; WO 87/00195; U.S. Pat. Re. 30,985;or U.S. Pat. No. 5,122,469, the disclosures of all of which areincorporated herein by reference, may be used as culture media for thehost cells. Any of these media may be supplemented as necessary withhormones and/or other growth factors (such as insulin, transferrin, orepidermal growth factor), salts (such as sodium chloride, calcium,magnesium, and phosphate), buffers (such as HEPES), nucleosides (such asadenosine and thymidine), antibiotics (such as Gentamycin™ drug), traceelements (defined as inorganic compounds usually present at finalconcentrations in the micromolar range), and glucose or an equivalentenergy source. Any other necessary supplements may also be included atappropriate concentrations that would be known to those skilled in theart. The culture conditions, such as temperature, pH, and the like, arethose previously used with the host cell selected for expression, andwill be apparent to the ordinarily skilled artisan.

Temporally Separate Expression of Light and Heavy Chains

Once the host cells are grown to a certain density, the culturingconditions are modified to promote the synthesis of the protein(s).According to the present invention, the light and heavy chains areinduced at different time during the synthesis phase. In one aspect, thetemporally separate expression of light and heavy chains is realized byusing a dual-promoter vector as described above. If induciblepromoter(s) are used in the dual-promoter vector of the invention,protein expression is induced under conditions suitable for theactivation of the promoter. In a preferred embodiment, both promotersare inducible. More preferably, the dual promoters are phoA and TacII,respectively. For example, a vector can be made wherein a phoA promoteris used for controlling transcription of the light chain, and a TacIIpromoter is used for controlling transcription of the heavy chain.During the first stage of induction, prokaryotic host cells transformedwith such a phoA/TacII dual promoter vector are cultured in aphosphate-limiting medium for the induction of the phoA promoter and theexpression of the light chain. After a desired period of time for lightchain expression, sufficient amount of IPTG is added to the culture forthe induction of the TacII promoter and the production of the heavychain.

In one aspect, the antibody or antibody fragment of the invention can beexpressed in the cytoplasm of a host bacteria cells. Various methods canbe used to improve production of soluble and functional antibodies orantibody fragments in E. coli cytoplasm. For example, E. coli strainsdeficient in the trxB gene have been found to enhance the formation ofdisulfide bonds in the cytoplasm and therefore useful for promotingexpression of functional antibody molecules with proper disulfide bondformations in the cytoplasm. Proba et al. (1995) Gene 159:203-207.Antibody fragment variants can be made to replace cysteine residues suchthat the variant does not require formation of disulfide bonds in bothV_(H) and V_(L), such antibody fragment variants, sometime referred toas “intrabodies”, can therefore be made in a reducing environment thatis not compatible with efficient disulfide bridge formation, such as inbacteria cytoplasm. Proba et al. (1998) J. Mol. Biol. 275:245-253.

When secretion signal sequences are used in the vector of the invention,the expressed light and heavy chain polypeptides are secreted into, andrecovered from, the periplasm of the host cells. Protein recoverytypically involves disrupting the microorganism, generally by such meansas osmotic shock, sonication or lysis. Once cells are disrupted, celldebris or whole cells may be removed by centrifugation or filtration.The proteins may be further purified, for example, by affinity resinchromatography. Alternatively, proteins can be transported into theculture media and isolated therein. Cells may be removed from theculture and the culture supernatant being filtered and concentrated forfurther purification of the proteins produced. The expressedpolypeptides can be further isolated and identified using commonly knownmethods such as polyacrylamide gel electrophoresis (PAGE) and Westernblot assay.

In one aspect of the invention, the antibody is produced in largequantity by a fermentation process. Various large-scale fed-batchfermentation procedures are available for production of recombinantproteins. Large-scale fermentations have at least 1000 liters ofcapacity, preferably about 1,000 to 100,000 liters of capacity. Thesefermentors use agitator impellers or other suitable means to distributeoxygen and nutrients, especially glucose (the preferred carbon/energysource). Small scale fermentation refers generally to fermentation in afermentor that is no more than approximately 100 liters in volumetriccapacity, and can range from about 1 liter to about 100 liters.

In a fermentation process, induction of protein expression is typicallyinitiated after the cells have been grown under suitable conditions to adesired density, e.g., an OD₅₅₀ of about 180-270. A variety of inducersmay be used, according to the vector construct employed, as is known inthe art and described above. Cells may be grown for shorter periodsprior to induction. Cells are usually induced for about 12-50 hours,although longer or shorter induction time may be used.

To further improve the production yield and quality of the antibodymolecules of the invention, various fermentation conditions can bemodified. For example, to improve the proper assembly and folding of thesecreted antibody polypeptides, additional vectors overexpressingchaperone proteins, such as Dsb proteins (DsbA, DsbB, DsbC, DsbD and orDsbG) or FkpA (a peptidylprolyl cis,trans-isomerase with chaperoneactivity) can be used to co-transform the host prokaryotic cells. Thechaperone proteins have been demonstrated to facilitate the properfolding and solubility of heterologous proteins produced in bacterialhost cells. Chen et al. (1999) J Bio Chem 274:19601-19605; Georgiou etal., U.S. Pat. No. 6,083,715; Georgiou et al., U.S. Pat. No. 6,027,888;Bothmann and Pluckthun (2000) J. Biol. Chem. 275:17100-17105; Ramm andPluckthun (2000) J. Biol. Chem. 275:17106-17113; Arie et al. (2001) Mol.Microbiol. 39:199-210. Sufficient disulfide bonds are particularlyimportant for the formation and folding of full length, bivalentantibodies having two heavy chains and two light chains.

To minimize proteolysis of expressed heterologous proteins (especiallythose that are proteolytically sensitive) such as in prokaryotic hostcells, certain host strains deficient for proteolytic enzymes can beused for the present invention. For example, prokaryotic host cellstrains may be modified to effect genetic mutation(s) in the genesencoding known bacterial proteases such as Protease III, OmpT, DegP,Tsp, Protease I, Protease Mi, Protease V, Protease VI and combinationsthereof. Some E. coli protease-deficient strains are available anddescribed in, for example, Joly et al. (1998), supra; Georgiou et al.,U.S. Pat. No. 5,264,365; Georgiou et al., U.S. Pat. No. 5,508,192; Haraet al. (1996) Microbial Drug Resistance 2:63-72.

Antibody Purification

Antibody compositions prepared from the host cells are preferablysubjected to at least one purification step. Examples of suitablepurification steps include hydroxylapatite chromatography, gelelectrophoresis, dialysis, and affinity chromatography, with affinitychromatography being the preferred purification technique. Thesuitability of particular protein as an affinity ligand depends on thespecies and isotype of any immunoglobulin Fc domain that is present inthe antibody. For example, protein A can be used to purify antibodiesthat are based on human γ1, γ2, or γ4 heavy chains. Lindmark et al.(1983) J. Immunol. Meth. 62:1-13. Protein G is recommended for all mouseisotypes and for human γ3 Guss et al. (1986) EMBO J. 5:15671575. Thematrix to which the affinity ligand is attached is most often agarose,but other matrices are available. Mechanically stable matrices such ascontrolled pore glass or poly(styrenedivinyl)benzene allow for fasterflow rates and shorter processing times than can be achieved withagarose. Where the antibody comprises a CH3 domain, the Bakerbond ABX™resin (J. T. Baker, Phillipsburg, N.J.) is useful for purification.Other techniques for protein purification such as fractionation on anion-exchange column, ethanol precipitation, Reverse Phase HPLC,chromatography on silica, chromatography on heparin SEPHAROSE™,chromatography on an anion or cation exchange resin (such as apolyaspartic acid column), chromatofocusing, SDS-PAGE, and ammoniumsulfate precipitation are also available depending on the antibody to berecovered.

In a preferred embodiment, the antibody produced herein is furtherpurified to obtain preparations that are more substantially homogeneousfor further assays and uses. For example, the hydrophobic interactionchromatography (HIC), particularly the low pH HIC (LPHIC) as describedin the U.S. Pat. No. 5,641,870, can be used for further purification. Inparticular, LPHIC provides a way to remove a correctly folded anddisulfide bonded antibody from unwanted contaminants (e.g., incorrectlyassociated light and heavy fragments).

Activity Assays

The antibody of the present invention can be characterized for itsphysical/chemical properties and biological functions by various assaysknown in the art. In one aspect of the invention, it is important tocompare the antibody made in the dual-promoter systems of the presentinvention to similar antibodies made in other expression systems, suchas different expression vector designs or different host cell systems.Particularly, the quantity of the assembled antibody complex expressedby the dual-promoter vector of the present invention can be compared tothose expressed by various polycistronic vectors. Methods for proteinquantification are well known in the art. For example, samples of theexpressed proteins can be compared for their quantitative intensities ona Coomassie-stained SDS-PAGE. Alternatively, the specific band(s) ofinterest (e.g., the assembled band) can be detected by, for example,western blot gel analysis.

The purified antibody can be further characterized by a series of assaysincluding, but not limited to, N-terminal sequencing, amino acidanalysis, non-denaturing size exclusion high pressure liquidchromatography (HPLC), mass spectrometry, ion exchange chromatographyand papain digestion.

In certain embodiments of the invention, the antibody produced herein isanalyzed for its biological activity. Preferably, the antibody of thepresent invention is tested for its antigen binding activity. Theantigen binding assays that are known in the art and can be used hereininclude without limitation any direct or competitive binding assaysusing techniques such as western blots, radioimmunoassays, ELISA (enzymelinked immunosorbent assay), “sandwich” immunoassays,immunoprecipitation assays, fluorescent immunoassays, and protein Aimmunoassays. An example of an antigen binding assay is provided belowin the Examples section.

Uses of the Antibody

An antibody of the present invention may be used, for example, topurify, detect, and target a specific polypeptide it recognizes,including both in vitro and in vivo diagnostic, prophylactic ortherapeutic methods for a variety of disorders or diseases.

A “disorder” is any condition that would benefit from treatment with theantibody. This includes chronic and acute disorders or diseasesincluding those pathological conditions which predispose the mammal tothe disorder in question. Non-limiting examples of disorders to betreated herein include malignant and benign tumors; non-leukemias andlymphoid malignancies; neuronal, glial, astrocytal, hypothalamic andother glandular, macrophagal, epithelial, stromal and blastocoelicdisorders; and inflammatory, angiogenic and immunologic disorders.

An “autoimmune disease” herein is a non-malignant disease or disorderarising from and directed against an individual's own tissues. Theautoimmune diseases herein specifically exclude malignant or cancerousdiseases or conditions, especially excluding B cell lymphoma, acutelymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), Hairycell leukemia and chronic myeloblastic leukemia. Examples of autoimmunediseases or disorders include, but are not limited to, inflammatoryresponses such as inflammatory skin diseases including psoriasis anddermatitis (e.g. atopic dermatitis); systemic scleroderma and sclerosis;responses associated with inflammatory bowel disease (such as Crohn'sdisease and ulcerative colitis); respiratory distress syndrome(including adult respiratory distress syndrome; ARDS); dermatitis;meningitis; encephalitis; uveitis; colitis; glomerulonephritis; allergicconditions such as eczema and asthma and other conditions involvinginfiltration of T cells and chronic inflammatory responses;atherosclerosis; leukocyte adhesion deficiency; rheumatoid arthritis;systemic lupus erythematosus (SLE); diabetes mellitus (e.g. Type Idiabetes mellitus or insulin dependent diabetes mellitis); multiplesclerosis; Reynaud's syndrome; autoimmune thyroiditis; allergicencephalomyelitis; Sjorgen's syndrome; juvenile onset diabetes; andimmune responses associated with acute and delayed hypersensitivitymediated by cytokines and T-lymphocytes typically found in tuberculosis,sarcoidosis, polymyositis, granulomatosis and vasculitis; perniciousanemia (Addison's disease); diseases involving leukocyte diapedesis;central nervous system (CNS) inflammatory disorder; multiple organinjury syndrome; hemolytic anemia (including, but not limited tocryoglobinemia or Coombs positive anemia); myasthenia gravis;antigen-antibody complex mediated diseases; anti-glomerular basementmembrane disease; antiphospholipid syndrome; allergic neuritis; Graves'disease; Lambert-Eaton myasthenic syndrome; pemphigoid bullous;pemphigus; autoimmune polyendocrinopathies; Reiter's disease; stiff-mansyndrome; Behcet disease; giant cell arteritis; immune complexnephritis; IgA nephropathy; IgM polyneuropathies; immunethrombocytopenic purpura (ITP) or autoimmune thrombocytopenia etc.

The terms “cancer” and “cancerous” refer to or describe thephysiological condition in mammals that is typically characterized byunregulated cell growth. Examples of cancer include but are not limitedto carcinoma, lymphoma, blastoma, sarcoma, and leukemia. More particularexamples of such cancers include squamous cell cancer, small-cell lungcancer, non-small cell lung cancer, adenocarcinoma of the lung, squamouscarcinoma of the lung, cancer of the peritoneum, hepatocellular cancer,gastrointestinal cancer, pancreatic cancer, glioblastoma, cervicalcancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breastcancer, colon cancer, colorectal cancer, endometrial or uterinecarcinoma, salivary gland carcinoma, kidney cancer, liver cancer,prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma andvarious types of head and neck cancer.

As used herein, “treatment” refers to clinical intervention in anattempt to alter the natural course of the individual or cell beingtreated, and can be performed either for prophylaxis or during thecourse of clinical pathology. Desirable effects of treatment includepreventing occurrence or recurrence of disease, alleviation of symptoms,diminishment of any direct or indirect pathological consequences of thedisease, preventing metastasis, decreasing the rate of diseaseprogression, amelioration or palliation of the disease state, andremission or improved prognosis.

An “effective amount” refers to an amount effective, at dosages and forperiods of time necessary, to achieve the desired therapeutic orprophylactic result. A “therapeutically effective amount” of theantibody may vary according to factors such as the disease state, age,sex, and weight of the individual, and the ability of the antibody toelicit a desired response in the individual. A therapeutically effectiveamount is also one in which any toxic or detrimental effects of theantibody are outweighed by the therapeutically beneficial effects. A“prophylactically effective amount” refers to an amount effective, atdosages and for periods of time necessary, to achieve the desiredprophylactic result. Typically, since a prophylactic dose is used insubjects prior to or at an earlier stage of disease, theprophylactically effective amount will be less than the therapeuticallyeffective amount.

In one aspect, an antibody of the invention can be used in immunoassaysfor qualitatively and quantitatively measuring specific antigens inbiological samples. Conventional methods for detecting antigen-antibodybinding includes, for example, an enzyme linked immunosorbent assay(ELISA), an radioimmunoassay (RIA) or tissue immunohistochemistry. Manymethods may use a label bound to the antibody for detection purposes.The label used with the antibody is any detectable functionality thatdoes not interfere with its binding to antibody. Numerous labels areknown, including the radioisotopes ³²P, ³²S, ¹⁴C, ¹²⁵I, ³H, and ¹³¹I,fluorophores such as rare earth chelates or fluorescein and itsderivatives, rhodamine and its derivatives, dansyl, umbelliferone,luceriferases, e.g., firefly luciferase and bacterial luciferase (U.S.Pat. No. 4,737,456), luciferin, 2,3-dihydrophthalazinediones,horseradish peroxidase (HRP), alkaline phosphatase,.beta.-galactosidase, glucoamylase, lysozyme, saccharide oxidases, e.g.,glucose oxidase, galactose oxidase, and glucose-6-phosphatedehydrogenase, heterocyclic oxidases such as uricase and xanthineoxidase, lactoperoxidase, biotin/avidin, spin labels, bacteriophagelabels, stable free radicals, imaging radionuclides (such as Technecium)and the like.

Conventional methods are available to bind these labels covalently tothe antibody polypeptides. For instance, coupling agents such asdialdehydes, carbodiimides, dimaleimides, bis-imidates, bis-diazotizedbenzidine, and the like may be used to tag the antibodies with theabove-described fluorescent, chemiluminescent, and enzyme labels. See,for example, U.S. Pat. No. 3,940,475 (fluorimetry) and U.S. Pat. No.3,645,090 (enzymes); Hunter et al. Nature 144: 945 (1962); David et al.Biochemistry 13:1014-1021 (1974); Pain et al. J. Immunol. Methods40:219-230 (1981); and Nygren Histochem, and Cytochem 30:407-412 (1982).Preferred labels herein are enzymes such as horseradish peroxidase andalkaline phosphatase. The conjugation of such label, including theenzymes, to the antibody -polypeptide is a standard manipulativeprocedure for one of ordinary skill in immunoassay techniques. See, forexample, O'Sullivan et al., “Methods for the Preparation ofEnzyme-antibody Conjugates for Use in Enzyme Immunoassay,” in Methods inEnzymology, ed. J. J. Langone and H. Van Vunakis, Vol. 73 (AcademicPress, New York, N.Y., 1981), pp. 147-166. Such bonding methods aresuitable for use with the antibody polypeptides of this invention.

Alternative to labeling the antibody, antigen can be assayed inbiological fluids by a competition immunoassay utilizing a competingantigen standard labeled with a detectable substance and an unlabeledantibody. In this assay, the biological sample, the labeled antigenstandards and the antibody are combined and the amount of labeledantigen standard bound to the unlabeled antibody is determined. Theamount of tested antigen in the biological sample is inverselyproportional to the amount of labeled antigen standard bound to theantibody.

In one aspect, the antibody of the invention is particularly useful todetect and profile expressions of specific surface antigens in vitro orin vivo. The surface antigen can be specific to a particular cell ortissue type, therefore serving as a marker of the cell or tissue type.Preferably, the surface antigen marker is differentially expressed atvarious differentiation stages of particular cell or tissue types. Theantibody directed against such surface antigen can thus be used for thescreening of cell or tissue populations expressing the marker. Forexample, the antibody of the invention can be used for the screening andisolation of stem cells such as embryonic stem cells, hematopoietic stemcells and mesenchymal stem cells. The antibody of the invention can alsobe used to detect tumor cells expressing tumor-associated surfaceantigens such HER2, HER3 or HER4 receptors.

The antibody of the invention may be used as an affinity purificationagent. In this process, the antibody is immobilized on a solid phasesuch a Sephadex resin or filter paper, using methods well known in theart. The immobilized antibody is contacted with a sample containing theantigen to be purified, and thereafter the support is washed with asuitable solvent that will remove substantially all the material in thesample except the antigen to be purified, which is bound to theimmobilized antibody. Finally, the support is washed with anothersuitable solvent, such as glycine buffer, pH 5.0, that will release theantigen from the antibody.

The antibody of the invention can be used as an antagonist to partiallyor fully block the specific antigen activity both in vitro and in vivo.Moreover, at least some of the antibodies of the invention canneutralize antigen activity from other species. Accordingly, theantibodies of the invention can be used to inhibit a specific antigenactivity, e.g., in a cell culture containing the antigen, in humansubjects or in other mammalian subjects having the antigen with which anantibody of the invention cross-reacts (e.g. chimpanzee, baboon,marmoset, cynomolgus and rhesus, pig or mouse).

In another embodiment, an antibody of the invention can be used in amethod for inhibiting an antigen in a subject suffering from a disorderin which the antigen activity is detrimental, comprising administeringto the subject an antibody of the invention such that the antigenactivity in the subject is inhibited. Preferably, the antigen is a humanprotein molecule and the subject is a human subject. Alternatively, thesubject can be a mammal expressing the antigen with which an antibody ofthe invention binds. Still further the subject can be a mammal intowhich the antigen has been introduced (e.g., by administration of theantigen or by expression of an antigen transgene). An antibody of theinvention can be administered to a human subject for therapeuticpurposes. Moreover, an antibody of the invention can be administered toa non-human mammal expressing an antigen with which the antibodycross-reacts (e.g., a primate, pig or mouse) for veterinary purposes oras an animal model of human disease. Regarding the latter, such animalmodels may be useful for evaluating the therapeutic efficacy ofantibodies of the invention (e.g., testing of dosages and time coursesof administration). Blocking antibodies of the invention that aretherapeutically useful include, for example but not limited to,anti-VEGF, anti-IgE, anti-CD11 and anti-tissue factor antibodies. Theblocking antibodies of the invention can be used to diagnose, treat,inhibit or prevent diseases, disorders or conditions associated withabnormal expression and or activity of one or more antigen molecules,including but not limited to malignant and benign tumors; non-leukemiasand lymphoid malignancies; neuronal, glial, astrocytal, hypothalamic andother glandular, macrophagal, epithelial, stromal and blastocoelicdisorders; and inflammatory, angiogenic and immunologic disorders.

In one aspect, the blocking antibody of the invention is specific to aligand antigen, and inhibits the antigen activity by blocking orinterfering with the ligand-receptor interaction involving the ligandantigen, thereby inhibiting the corresponding signal pathway and othermolecular or cellular events. The invention also featuresreceptor-specific antibodies which do not necessarily prevent ligandbinding but interfere with receptor activation, thereby inhibiting anyresponses that would normally be initiated by the ligand binding. Theinvention also encompasses antibodies that either preferably orexclusively bind to ligand-receptor complexes. The antibody of theinvention can also act as an agonist of a particular antigen receptor,thereby potentiating, enhancing or activating either all or partialactivities of the ligand-mediated receptor activation.

In certain embodiments, an immunoconjugate comprising the antibodyconjugated with a cytotoxic agent is made and used. Preferably, theimmunoconjugate and/or antigen to which it is bound is/are internalizedby the cell, resulting in increased therapeutic efficacy of theimmunoconjugate in killing the target cell to which it binds. In apreferred embodiment, the cytotoxic agent targets or interferes withnucleic acid in the target cell.

The term “cytotoxic agent” as used herein refers to a substance thatinhibits or prevents the function of cells and/or causes destruction ofcells. The term is intended to include radioactive isotopes (e.g. At²¹¹,I¹³¹, I¹²⁵, Y⁹⁰ Re¹⁸⁶, Re¹⁸⁸, Sm¹⁵³, Bi²¹², P³² and radioactive isotopesof Lu), chemotherapeutic agents, and toxins such as small moleculetoxins or enzymatically active toxins of bacterial, fungal, plant oranimal origin, including fragments and/or variants thereof.

A “chemotherapeutic agent” is a chemical compound useful in thetreatment of cancer. Examples of chemotherapeutic agents includealkylating agents such as thiotepa and cyclosphosphamide (CYTOXAN™);alkyl sulfonates such as busulfan, improsulfan and piposulfan;aziridines such as benzodopa, carboquone, meturedopa, and uredopa;ethylenimines and methylamelamines including altretamine,triethylenemelamine, trietylenephosphoramide,triethylenethiophosphaoramide and trimethylolomelamine; acetogenins(especially bullatacin and bullatacinone); a camptothecin (including thesynthetic analogue topotecan); bryostatin; callystatin; CC-1065(including its adozelesin, carzelesin and bizelesin syntheticanalogues); cryptophycins (particularly cryptophycin 1 and cryptophycin8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189and CBI-TMI); eleutherobin; pancratistatin; a sarcodictyin;spongistatin; nitrogen mustards such as chlorambucil, chlornaphazine,cholophosphamide, estramustine, ifosfamide, mechlorethamine,mechlorethamine oxide hydrochloride, melphalan, novembichin,phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureassuch as carmustine, chlorozotocin, fotemustine, lomustine, nimustine,ranimustine; antibiotics such as the enediyne antibiotics (e.g.calicheamicin, especially calicheamicin γ₁ ¹ and calicheamicin θ¹ ₁),see, e.g., Agnew Chem. Intl. Ed Engl. 33:183-186 (1994); dynemicin,including dynemicin A; an esperamicin; as well as neocarzinostatinchromophore and related chromoprotein enediyne antiobioticchromomophores), aclacinomysins, actinomycin, authramycin, azaserine,bleomycins, cactinomycin, carabicin, caminomycin, carzinophilin,chromomycins, dactinomycin, daunorubicin, detorubicin,6-diazo-5-oxo-L-norleucine, doxorubicin (includingmorpholino-doxorubicin, cyanomorpholino-doxorubicin,2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin,idarubicin, marcellomycin, mitomycins, mycophenolic acid, nogalamycin,olivomycins, peplomycin, potfiromycin, puromycin, quelamycin,rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex,zinostatin, zorubicin; anti-metabolites such as methotrexate and5-fluorouracil (5-FU); folic acid analogues such as denopterin,methotrexate, pteropterin, trimetrexate; purine analogs such asfludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidineanalogs such as ancitabine, azacitidine, 6-azauridine, carmofur,cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine,5-FU; androgens such as calusterone, dromostanolone propionate,epitiostanol, mepitiostane, testolactone; anti-adrenals such asaminoglutethimide, mitotane, trilostane; folic acid replenisher such asfrolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinicacid; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine;demecolcine; diaziquone; elfomithine; elliptinium acetate; anepothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan;lonidamine; maytansinoids such as maytansine and ansamitocins;mitoguazone; mitoxantrone; mopidamol; nitracrine; pentostatin; phenamet;pirarubicin; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK®;razoxane; rhizoxin; sizofuran; spirogermanium; tenuazonic acid;triaziquone; 2,2′,2″-trichlorotriethylamine; trichothecenes (especiallyT-2 toxin, verracurin A, roridin A and anguidine); urethan; vindesine;dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman;gacytosine; arabinoside (“Ara-C”); cyclophosphamide; thiotepa; taxoids,e.g. paclitaxel (TAXOL®, Bristol-Myers Squibb Oncology, Princeton, N.J.)and doxetaxel (TAXOTERE®, Rhône-Poulenc Rorer, Antony, France);chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate;platinum analogs such as cisplatin and carboplatin; vinblastine;platinum; etoposide (VP-16); ifosfamide; mitomycin C; mitoxantrone;vincristine; vinorelbine; navelbine; novantrone; teniposide; daunomycin;aminopterin; xeloda; ibandronate; CPT-11; topoisomerase inhibitor RFS2000; difluoromethylornithine (DMFO); retinoic acid; capecitabine; andpharmaceutically acceptable salts, acids or derivatives of any of theabove. Also included in this definition are anti-hormonal agents thatact to regulate or inhibit hormone action on tumors such asanti-estrogens including for example tamoxifen, raloxifene, aromataseinhibiting 4(5) -imidazoles, 4-hydroxytamoxifen, trioxifene, keoxifene,LY117018, onapristone, and toremifene (Fareston); and anti-androgenssuch as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin:and pharmaceutically acceptable salts, acids or derivatives of any ofthe above.

Conjugates of an antibody and one or more small molecule toxins, such asa calicheamicin, a maytansine (U.S. Pat. No. 5,208,020), a trichothene,and CC1065 are also contemplated herein.

In one preferred embodiment of the invention, the antibody is conjugatedto one or more maytansine molecules (e.g. about 1 to about 10 maytansinemolecules per antibody molecule). Maytansine may, for example, beconverted to May-SS-Me which may be reduced to May-SH3 and reacted withmodified antibody (Chari et al. Cancer Research 52: 127-131 (1992)) togenerate a maytansinoid-antibody immunoconjugate.

Another immunoconjugate of interest comprises an antibody conjugated toone or more calicheamicin molecules. The calicheamicin family ofantibiotics are capable of producing double-stranded DNA breaks atsub-picomolar concentrations. Structural analogues of calicheamicinwhich may be used include, but are not limited to, γ₁ ¹, α₂ ¹, α₃ ¹,N-acetyl-γ₁ ¹, PSAG and θ¹ ₁ (Hinman et al. Cancer Research 53:3336-3342 (1993) and Lode et al. Cancer Research 58: 2925-2928 (1998)).See, also, U.S. Pat. Nos. 5,714,586; 5,712,374; 5,264,586; and5,773,001.

Enzymatically active toxins and fragments thereof which can be usedinclude diphtheria A chain, nonbinding active fragments of diphtheriatoxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain,abrin A chain, modeccin A chain, alpha-sarcin, Aleuritesfordii proteins,dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, andPAP-S), momordica charantia inhibitor, curcin, crotin, sapaonariaofficinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin,enomycin and the tricothecenes. See, for example, WO 93/21232 publishedOct. 28, 1993.

The present invention further contemplates an immunoconjugate formedbetween an antibody and a compound with nucleolytic activity (e.g. aribonuclease or a DNA endonuclease such as a deoxyribonuclease; DNase).

A variety of radioactive isotopes are available for the production ofradioconjugated antibodies. Examples include At²¹¹, I¹³¹, I¹²⁵, Y⁹⁰,Re¹⁸⁶, Re¹⁸⁸, Sm¹⁵³, Bi²¹², P³² and radioactive isotopes of Lu.

Conjugates of the antibody and cytotoxic agent may be made using avariety of bifunctional protein coupling agents such asN-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP),succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate,iminothiolane (IT), bifunctional derivatives of imidoesters (such asdimethyl adipimidate HCL), active esters (such as disuccinimidylsuberate), aldehydes (such as glutareldehyde), bis-azido compounds (suchas bis(p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (suchas bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astolyene 2,6-diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin canbe prepared as described in Vitetta et al. Science 238: 1098 (1987).Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent forconjugation of radionucleotide to the antibody. See WO94/11026. Thelinker may be a “cleavable linker” facilitating release of the cytotoxicdrug in the cell. For example, an acid-labile linker,peptidase-sensitive linker, dimethyl linker or disulfide-containinglinker (Chari et al. Cancer Research 52: 127-131 (1992)) may be used.

Alternatively, a fusion protein comprising the antibody and cytotoxicagent may be made, e.g. by recombinant techniques or peptide synthesis.

In yet another embodiment, the antibody may be conjugated to a“receptor” (such as streptavidin) for utilization in tumor pretargetingwherein the antibody-receptor conjugate is administered to the patient,followed by removal of unbound conjugate from the circulation using aclearing agent and then administration of a “ligand” (e.g. avidin) whichis conjugated to a cytotoxic agent (e.g. a radionucleotide).

Antibodies of the present invention can be used either alone or incombination with other compositions in a therapy. For instance, theantibody may be co-administered with another antibody, chemotherapeuticagent(s) (including cocktails of chemotherapeutic agents), othercytotoxic agent(s), anti-angiogenic agent(s), cytokines, and/or growthinhibitory agent(s). Where the antibody inhibits tumor growth, it may beparticularly desirable to combine the antibody with one or more othertherapeutic agent(s) which also inhibits tumor growth. For instance,anti-VEGF antibodies blocking VEGF activities may be combined withanti-ErbB antibodies (e.g. HERCEPTIN® anti-HER2 antibody) in a treatmentof metastatic breast cancer. Alternatively, or additionally, the patientmay receive combined radiation therapy (e.g. external beam irradiationor therapy with a radioactive labelled agent, such as an antibody). Suchcombined therapies noted above include combined administration (wherethe two or more agents are included in the same or separateformulations), and separate administration, in which case,administration of the antibody can occur prior to, and/or following,administration of the adjunct therapy or therapies.

The antibody (and adjunct therapeutic agent) is/are administered by anysuitable means, including parenteral, subcutaneous, intraperitoneal,intrapulmonary, and intranasal, and, if desired for local treatment,intralesional administration. Parenteral infusions includeintramuscular, intravenous, intraarterial, intraperitoneal, orsubcutaneous administration. In addition, the antibody is suitablyadministered by pulse infusion, particularly with declining doses of theantibody. Preferably the dosing is given by injections, most preferablyintravenous or subcutaneous injections, depending in part on whether theadministration is brief or chronic.

The antibody composition of the invention will be formulated, dosed, andadministered in a fashion consistent with good medical practice. Factorsfor consideration in this context include the particular disorder beingtreated, the particular mammal being treated, the clinical condition ofthe individual patient, the cause of the disorder, the site of deliveryof the agent, the method of administration, the scheduling ofadministration, and other factors known to medical practitioners. Theantibody need not be, but is optionally formulated with one or moreagents currently used to prevent or treat the disorder in question. Theeffective amount of such other agents depends on the amount of antibodypresent in the formulation, the type of disorder or treatment, and otherfactors discussed above. These are generally used in the same dosagesand with administration routes as used hereinbefore or about from 1 to99% of the heretofore employed dosages.

For the prevention or treatment of disease, the appropriate dosage ofthe antibody (when used alone or in combination with other agents suchas chemotherapeutic agents) will depend on the type of disease to betreated, the type of antibody, the severity and course of the disease,whether the antibody is administered for preventive or therapeuticpurposes, previous therapy, the patient's clinical history and responseto the antibody, and the discretion of the attending physician. Theantibody is suitably administered to the patient at one time or over aseries of treatments. Depending on the type and severity of the disease,about 1 μg/kg to 15 mg/kg (e.g. 0.1 mg/kg-10 mg/kg) of antibody is aninitial candidate dosage for administration to the patient, whether, forexample, by one or more separate administrations, or by continuousinfusion. A typical daily dosage might range from about 1 μg/kg to 100mg/kg or more, depending on the factors mentioned above. For repeatedadministrations over several days or longer, depending on the condition,the treatment is sustained until a desired suppression of diseasesymptoms occurs. The preferred dosage of the antibody will be in therange from about 0.05 mg/kg to about 10 mg/kg. Thus, one or more dosesof about 0.5 mg/kg, 2.0 mg/kg, 4.0 mg/kg or 10 mg/kg (or any combinationthereof) may be administered to the patient. Such doses may beadministered intermittently, e.g. every week or every three weeks (e.g.such that the patient receives from about two to about twenty, e.g.about six doses of the antibody). An initial higher loading dose,followed by one or more lower doses may be administered. An exemplarydosing regimen comprises administering an initial loading dose of about4 mg/kg, followed by a weekly maintenance dose of about 2 mg/kg of theantibody. However, other dosage regimens may be useful. The progress ofthis therapy is easily monitored by conventional techniques and assays.

Pharmaceutical Formulations

Therapeutic formulations of the antibody are prepared for storage bymixing the antibody having the desired degree of purity with optionalphysiologically acceptable carriers, excipients or stabilizers(Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)),in the form of aqueous solutions, lyophilized or other driedformulations. Acceptable carriers, excipients, or stabilizers arenontoxic to recipients at the dosages and concentrations employed, andinclude buffers such as phosphate, citrate, histidine and other organicacids; antioxidants including ascorbic acid and methionine;preservatives (such as octadecyldimethylbenzyl ammonium chloride;hexamethonium chloride; benzalkonium chloride, benzethonium chloride;phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol);low molecular weight (less than about 10 residues) polypeptides;proteins, such as serum albumin, gelatin, or immunoglobulins;hydrophilic polymers such as polyvinylpyrrolidone; amino acids such asglycine, glutamine, asparagine, histidine, arginine, or lysine;monosaccharides, disaccharides, and other carbohydrates includingglucose, mannose, or dextrins; chelating agents such as EDTA; sugarssuch as sucrose, mannitol, trehalose or sorbitol; salt-formingcounter-ions such as sodium; metal complexes (e.g., Zn-proteincomplexes); and/or non-ionic surfactants such as TWEEN™, PLURONICS™ orpolyethylene glycol (PEG).

The formulation herein may also contain more than one active compound asnecessary for the particular indication being treated, preferably thosewith complementary activities that do not adversely affect each other.Such molecules are suitably present in combination in amounts that areeffective for the purpose intended.

The active ingredients may also be entrapped in microcapsule prepared,for example, by coacervation techniques or by interfacialpolymerization, for example, hydroxymethylcellulose orgelatin-microcapsule and poly-(methylmethacylate) microcapsule,respectively, in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles andnanocapsules) or in macroemulsions. Such techniques are disclosed inRemington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

The formulations to be used for in vivo administration must be sterile.This is readily accomplished by filtration through sterile filtrationmembranes.

Sustained-release preparations may be prepared. Suitable examples ofsustained-release preparations include semipermeable matrices of solidhydrophobic polymers containing the antibody, which matrices are in theform of shaped articles, e.g., films, or microcapsule. Examples ofsustained-release matrices include polyesters, hydrogels (for example,poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides(U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and γethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradablelactic acid-glycolic acid copolymers such as the LUPRON DEPOT™(injectable microspheres composed of lactic acid-glycolic acid copolymerand leuprolide acetate), and poly-D-(−)-3-hydroxybutyric acid. Whilepolymers such as ethylene-vinyl acetate and lactic acid-glycolic acidenable release of molecules for over 100 days, certain hydrogels releaseproteins for shorter time periods. When encapsulated antibodies remainin the body for a long time, they may denature or aggregate as a resultof exposure to moisture at 37° C., resulting in a loss of biologicalactivity and possible changes in immunogenicity. Rational strategies canbe devised for stabilization depending on the mechanism involved. Forexample, if the aggregation mechanism is discovered to be intermolecularS—S bond formation through thio-disulfide interchange, stabilization maybe achieved by modifying sulfhydryl residues, lyophilizing from acidicsolutions, controlling moisture content, using appropriate additives,and developing specific polymer matrix compositions.

Articles of Manufacture

In another embodiment of the invention, an article of manufacturecontaining materials useful for the treatment of the disorders describedabove is provided. The article of manufacture comprises a container anda label or package insert on or associated with the container. Suitablecontainers include, for example, bottles, vials, syringes, etc. Thecontainers may be formed from a variety of materials such as glass orplastic. The container holds a composition which is effective fortreating the condition and may have a sterile access port (for examplethe container may be an intravenous solution bag or a vial having astopper pierceable by a hypodermic injection needle). At least oneactive agent in the composition is an antibody of the invention. Thelabel or package insert indicates that the composition is used fortreating the condition of choice, such as cancer. Moreover, the articleof manufacture may comprise (a) a first container with a compositioncontained therein, wherein the composition comprises a antibody; and (b)a second container with a composition contained therein, wherein thecomposition comprises a further cytotoxic agent. The article ofmanufacture in this embodiment of the invention may further comprise apackage insert indicating that the first and second antibodycompositions can be used to treat cancer. Alternatively, oradditionally, the article of manufacture may further comprise a second(or third) container comprising a pharmaceutically-acceptable buffer,such as bacteriostatic water for injection (BWFI), phosphate-bufferedsaline, Ringer's solution and dextrose solution. It may further includeother materials desirable from a commercial and user standpoint,including other buffers, diluents, filters, needles, and syringes.

The following examples are intended merely to illustrate the practice ofthe present invention and are not provided by way of limitation. Thedisclosures of all patent and scientific literatures cited herein areexpressly incorporated in their entirety by reference.

EXAMPLES Example 1 Production of antiCD18 Antibody Fragments

Materials & Methods

Plasmid Construction

The control plasmid, pS1130, was designed for the dicistronic expressionof anti-CD18 F(ab′)₂ and it was based on the vector described by Carteret al. (1992) Bio/Technology 10:163-167. This design placestranscription of the genes for both light chain and the heavy chainfragment with a C-terminal leucine zipper under the control of a singlephoA promoter. Transcription ends with a λt₀ transcriptional terminatorlocated downstream of the coding sequence for the heavy chain-leucinezipper (Scholtissek and Grosse (1987) Nucleic Acids Res. 15(7): 3185).The heat stable enterotoxin II signal sequence (STII) precedes thecoding sequence for each chain and directs the secretion of thepolypeptide into the periplasm (Lee et al. (1983) Infect. Immun. 42:264-268; Picken et al. (1983) Infect. Immun. 42: 269-275). Leucinezipper was attached to the C-terminal end of heavy chain fragment topromote the dimerization of the two Fab′ arms.

The dual-promoter plasmid containing two separate translational units,pxCD18-7T3, temporally separates the transcription of light chain fromthe transcription of heavy chain. As in pS1130, light chain remainsunder the control of the phoA promoter. However, in pxCD18-7T3, a λt₀ totranscriptional terminator follows the light chain coding sequence.Downstream of this terminator, the TacII promoter was added to controlthe transcription of the heavy chain fragment/C-terminal leucine zipper(DeBoer, et al. (1983) Proc. Natl. Acad. Sci. USA 80:21-25). A secondλt₀ transcriptional terminator follows this coding sequence. Silentcodon variants of the STII signal sequence were used to direct thesecretion of both chains (Simmons and Yansura (1996) NatureBiotechnology 14:629-634).

A schematic comparison of the single promoter control plasmid vs. thedual-promoter plasmid is depicted in FIG. 1. The expression cassettesequence of pxCD18-7T3 is provided in FIG. 2 (SEQ ID NO:3) and the aminoacid sequences from the two translational units are shown in FIG. 3 (SEQID NO:4).

Fermentation

The host strain used in fermentation was a derivative of E. coli W3110,designated 59A7. The complete genotype of 59A7 is W3110 ΔfhuA phoAΔE15Δ(argF-lac)169 deoC2 degP41(ΔpstI-Kan^(r)) IN(rrnD-rrnE) 1ilvG2096(Val^(r)) Δprc pre-suppressor. The 59A7 host cells weretransformed with either pS1130 or pxCD18-7T3 plasmid and successfultransformants were selected and grown in culture. In the case of thedual-promoter plasmid, an additional plasmid, pMS421, was co-transformedalong with pxCD18-7T3. This additional plasmid, pMS421, is apSC101-based plasmid which provides lacIq to improve control of theTacII promoter, and which also confers spectinomycin and streptomycinresistance.

For each 10-liter fermentation, a single vial containing 1.5 ml ofculture in 10-15% DMSO was thawed into a IL shake flask containing 500ml of LB medium supplemented with 0.5 ml of tetracycline solution (5mg/ml) and 2.5 ml 1M sodium phosphate solution. This seed culture wasgrown for approximately 16 hours at 30° C. and was then used toinoculate a 10-liter fermentor.

The fermentor initially started with approximately 6.5 liters of mediumcontaining about 4.4 g of glucose, 100 ml of 1M magnesium sulfate, 10 mlof a trace element solution (100 ml hydrochloric acid, 27 g ferricchloride hexahydrate, 8 g zinc sulfate heptahydrate, 7 g cobalt chloridehexahydrate, 7 g sodium molybdate dihydrate, 8 g cupric sulfatepentahydrate, 2 g boric acid, 5 g manganese sulfate monohydrate, in afinal volume of 1 liter), 20 ml of a tetracycline solution (5 mg/ml inethanol), 10 ml of Fermax Adjuvant 27 (or some equivalent anti-foam), 1bag of HCD salts (37.5 g ammonium sulfate, 19.5 g potassium phosphatedibasic, 9.75 g sodium phosphate monobasic dihydrate, 7.5 g sodiumcitrate dihydrate, 11.3 g potassium phosphate monobasic), and 200 g ofNZ Amine A (a protein hydrolysate). Fermentations were performed at 30°C. with 10 slpm of air flow and were controlled at a pH of 7.0±0.2(although occasional excursions beyond this range occurred in somecases). The back pressure of the fermentor and agitation rate werevaried to manipulate the oxygen transfer rate in the fermentor, and,consequently, control the cellular respiration rate.

Following inoculation of the fermentor with the cell-containing mediumfrom the shake flask, the culture was grown in the fermentor to highcell densities using a computer-based algorithm to feed a concentratedglucose solution to the fermentor. Ammonium hydroxide (58% solution) andsulfuric acid (24% solution) were also fed to the fermentor as needed tocontrol pH. Further additions of anti-foam were also used in some casesto control foaming. When the culture reached a cell density ofapproximately 40 OD550, an additional 100 ml of 1M magnesium sulfate wasadded to the fermentor. Additionally, a concentrated salt feed(consisting of approximately 10 g ammonium sulfate, 26 g dibasicpotassium phosphate, 13 g monobasic sodium phosphate dihydrate, 2 gsodium citrate dihydrate and 15 g monobasic potassium phosphate in 1 Lof water) to the fermentor was started at a rate of 2.5 ml/min when theculture reached approximately 20 OD550 and continued until approximately1250 ml were added to the fermentation. Fermentations were typicallycontinued for 72-80 hours.

During the fermentation, once the dissolved oxygen setpoint for thefermentation was reached, the concentrated glucose solution was fedbased on the dissolved oxygen probe signal in order to control thedissolved oxygen concentration at the setpoint. Consequently, in thiscontrol scheme, manipulations of fermentor operating parameters such asthe agitation rate or back pressure, which affect the oxygen transfercapacity in the fermentation, correspondingly manipulated the oxygenuptake rate or metabolic rate of the cells.

A mass spectrometer was used to monitor the composition of the off-gasfrom the fermentations and enabled the calculation of the oxygen uptakeand carbon dioxide evolution rates in the fermentations.

When the culture reached a cell density of approximately 220 OD550, theagitation was decreased from an initial rate of 1000 rpm toapproximately 725 rpm over approximately 12 hours. For the fermentationof the pxCD18-7T3 system (wherein the TacII promoter was used to controlheavy chain expression), 50 ml of 200 mM IPTG was added to induce heavychain synthesis approximately 12 hours after the culture reached a celldensity of 2200D550.

Product Assays

To assess the quantity of the antibody fragments produced in thefermentations, a number of protein assays were used. To determine thequantity of the assembled anti-CD18 F(ab′)₂-leucine zipper complex, aprotein G assay was used. Derrich and Wigley (1992) Nature 359:752-4. Toprepare samples for this assay, whole fermentation broth was firstsonicated and diluted 2.4× with 50 mM magnesium sulfate.Polyethyleneimine (PEI) was added to a final concentration of 0.1%.After a 20 minute incubation, the samples were centrifuged forapproximately 20 minutes at approximately 14,000×g in a microfuge. Thesupernatant was then diluted 2× with phosphate buffered saline andloaded on a protein G column (Poros G/M column) using a HP1090 or HP1100HPLC system. A 7 minute assay was used in which the column was firstequilibrated with 10 mM PO4/300 mM NaCl (pH 8). Following injection ofthe sample, the column was rinsed with the equilibration buffer forapproximately 5.5 minutes (using approx. 3 ml of buffer in a 2.1×30 mmcolumn), followed by a step elution using 30 mM PO4/150 mM NaCl/0.01%TFA (pH 1.9).

To confirm that the protein G results indeed represented assembledF(ab′)₂ complex, product was also purified through multiplechromatography steps including ion exchange and, after removal of theleucine zipper portion using immobilized pepsin, an additional, ionexchange column and a phenyl sepharose HIC column. Purified product wascharacterized by a number of routine methods, such as a cation exchangeassay, a capillary zone electrophoresis non-gel sieving assay, andSDS-PAGE gels.

To assess the total quantity of light chain and heavy chain fragmentsproduced in the fermentations, an alternative reversed-phase HPLC assaywas used. Samples used for this assay were prepared as described abovefor the protein G assay. The soluble lysate samples were diluted in 6MGuanidine-HCl, 50 mM TRIS, pH 9 (typically 100 μl of sample was dilutedwith 650 μl of the guanidine solution). 501l of 2M dithiothreitol(freshly thawed) was then added. Prior to loading on the HPLC, 200 μl ofacetonitrile was added and filtered through a 0.2 μm filter.

For the reversed-phase methodology, a Hewlett-Packard™ 1100 HPLC wasused with a Perseptive Poros™ R-1 reversed phase column. Analyses wererun with the column heated to 60-80° C. and UV absorbance at 278 nm wasmonitored. The column was equilibrated in a 28% acetonitrile solution inwater with 0.1% trifluoroacetic acid. 25 μl of sample was next loaded onthe column, and elution was performed using a linear gradient from 28%to 38% acetonitrile over 20 minutes followed by a 17 minute period ofregeneration at 95% acetonitrile and re-equilibration at 28%acetonitrile. Peaks for light chain and heavy chain-related species wereidentified by comparison with standards and analysis using massspectrometry for confirmation. Fermentation samples from a blank run inwhich the same host was used except with a plasmid not containing thesequences for heavy and light chain, were similarly prepared andanalyzed to determine the appropriate baselines for the analyses.Integration of the peak areas was performed using the Hewlett-Packard™1100 software and standards were spiked into blank run samples togenerate a calibration curve in order to quantify the relative quantityof the various species in the samples.

The insoluble lysate samples were also similarly analyzed byresuspending the insoluble pellets obtained from cell lysates in 950 μlof 6M Guanidine/HCl, 50 mM TRIS, pH 9+50 μl 2M dithiothreitol.Sonication (5 to 10 pulses) was typically performed to aid inresolubilizing the pellets followed by the dilution of 100 μl of theresuspended pellet in 650 μl of the guanidine solution+50 μl 2Mdithiothreitol+200 mM acetonitrile. The samples were then filtered andanalyzed using the same method as for the soluble lysate samples.

Results

A series of anti-CD18 F(ab′)₂ fermentation runs were conducted usingeither the pS 1130 single promoter system or the pxCD18-7T3dual-promoter system. The yields of assembled anti-CD18 F(ab′)₂ complexwere measured and calculated using the protein assays described in theMethods and Materials section. As shown in FIG. 4, which is a bar graphrepresenting the fermentation yields (g/L), use of the phoA-tacdual-promoter vector in the strain 59A7 increased yields of assembledF(ab′)₂ from approximately 2.5 g/L for the best pS1130/59A7 transformantidentified to approximately 4.6±0.5 g/L, a nearly two-fold, increase.

To further illustrate the improved properties of the dual-promotersystem, profiles of the expression of total heavy chain, soluble lightchain and assembled F(ab′)₂ complex were established for the singlepromoter system (pS1130/59A7) and the dual-promoter system(pxCD18-7T3/59A7), the results shown in FIGS. 5 and 6, respectively.Significantly, in the dual-promoter system wherein the light chain wasfirst expressed and secreted into the periplasmic space, followed by aprolonged period of heavy chain production, F(ab′)₂ assembly occurredalmost immediately following the induction of heavy chain expression(FIG. 6); whilst in the single promoter system, the initial F(ab′)₂assembly following the induction of both light and heavy chains wasrelatively poor (FIG. 5). Without intending to be bound by oneparticular theory, these results suggest that F(ab′)₂ assembly isinefficient until significant levels of soluble light chain accumulatedin the periplasm.

The two systems were further compared for their assembly efficiency,which is defined as the ratio of heavy chain in F(ab′)₂ complex to thetotal quantity of heavy chain synthesized. As shown in FIG. 7, thedual-promoter system provides an increased assembly efficiency comparedto the traditional single promoter system, particularly during theinitial period (first 10 hours) of heavy chain synthesis. A comparisonof the heavy chain expression levels of the two systems shows that useof the dual-promoter system also increased the total quantity of heavychain synthesized, as demonstrated in FIG. 7.

Therefore, the results show that by temporally separating the light andheavy chain synthesis, a significantly increased yield of assembledanti-CD18 F(ab′)₂ was obtained. The novel system and observations havebroad applications in other systems in which multiple protein units areto be expressed and assembled.

Example 2 Production of Anti-Tissue Factor IgG1

This example illustrates the continuing efforts in producing full lengthantibodies in an E. coli system. When both light and full-length heavychains were co-expressed simultaneously using strong TIR's, asignificant amount of expressed precursor polypeptides accumulated,resulting in less quantity of mature light and heavy chains and properlyassembled full length antibodies. This example shows that precursoraccumulation can be overcome by temporally separating the expression oflight and heavy chains. Placing each chain under the control of adifferent promoter averts the secretory block by allowing for theexpression of each chain at separate times. This approach permits theuse of stronger TIR's than can be used for simultaneous expression,potentially resulting in a higher level of secretion for each chain.Such expression constructions with higher expression level of individualchains are advantageous for improving yields of full length, properlyassembled antibodies.

Materials & Methods

Plasmid Construction

The expression cassette for the control plasmid, paTF130, comprises,from 5′ to 3′: (1) a phoA promoter (Kikuchi et al., Nucleic Acids Res.9(21):5671-5678 (1981)); (2) trp Shine-Dalgarno (Yanofsky et al.,Nucleic Acids Res. 9:6647-6668 (1981)); (3) a TIR variant of the STIIsignal sequence (TIR relative strength˜7) (Simmons and Yansura, NatureBiotechnology 14:629-634 (1996)); (4) coding sequence for anti-tissuefactor light chain; (5) λt₀ terminator (Scholtissek and Grosse, NucleicAcids Res. 15:3185 (1987)); (6) a second phoA promoter; (7) a second trpShine-Dalgarno; (8) a second silent codon variant of the STII signalsequence (TIR relative strength˜3); (9) coding sequence for anti-tissuefactor full-length heavy chain; and (10) a second λt₀ to terminator.This expression cassette was cloned into the framework of the E. coliplasmid pBR322. Sutcliffe (1978) Cold Spring Harbor Symp. Quant. Biol.43:77-90. Thus, the independent transcription of light chain from heavychain is achieved in this plasmid by placing each gene under the controlof its own phoA promoter; however, since both phoA promoters areinducible under identical conditions, both chains are expressedsimultaneously.

Alternatively, the vector design of pxTF-7T3FL allows for the temporallyseparate expression of each chain by using two different, rather thantwo identical, promoters. In this plasmid, light chain remains under thecontrol of the phoA promoter. However, the tacII promoter (DeBoer, et.al., Proc. Natl. Acad. Sci. USA 80:21-25 (1983)) is used to control thetranscription of heavy chain. As known in the art, phoA and tacIIpromoters are induced under substantially different conditions. Aschematic comparison of paTF130 and pxTF-7T3FL is depicted in FIG. 8.The nucleic acid sequence of pxTF-7T3FL and the polypeptide sequences itencodes are provided in FIG. 9 and FIG. 10, respectively.

Expression Induction, Sample Preparation and Analysis

For the small scale expression of each construct, E. coli strain 33D3,with genotype (W3110 kan^(R) ΔfhuA (ΔtonA) ptr3 phoAΔE15 lacIq lacL8ompT Δ(nmpc-fepE) deg P) was used as host cells. Followingtransformation, selected transformant picks were inoculated into 5 mlLuria-Bertani medium supplemented with carbenicillin (50 ug/ml) andgrown at 30° C. on a culture wheel overnight. Each culture was thendiluted (1:50) into C.R.A.P. phosphate-limiting media (3.57 g (NH4)₂SO4,0.71 g NaCitrate-2H₂O, 1.07 g KCl, 5.36 g Yeast Extract (certified),5.36 g HycaseSF-Sheffield, adjusted pH with KOH to 7.3, qs to 872 mlwith SQ H₂O and autoclaved; cooled to 55° C. and supplemented with 110ml 1M MOPS pH 7.3, 11 ml 50% glucose, 7 ml 1M MgSO4). Carbenicillin wasthen added to the induction culture at a concentration of 50 ug/ml and,unless otherwise noted, all shake flask inductions were performed in a 2ml volume.

Following inoculation into the induction medium, the conditions werevaried for each sample depending on the promoters used and the timing ofpromoter induction. For paTF130, the vector using phoA promoters tocontrol the transcription of both light and heavy chain genes, theinduction was carried out at 30° C. with shaking for ˜24 hours with noother additions made to the culture. Sufficient depletion of thephosphate in this sample leads to the simultaneous induction of the phoApromoters controlling both light and heavy chain transcription. ForpxTF-7T3FL, the vector using phoA promoter to control the light chainexpression but a tacII promoter to control the heavy chain expression,the induction was first carried out in the same culture conditions asused for paTF130. After 16 hours with shaking at 30° C., potassiumphosphate buffer (pH 7.4) was added to a final concentration of 1 mM.Approximately 45 minutes later, IPTG was added to the culture to a finalconcentration of 1 mM to induce the tacII promoter. The induction wasthen continued for another ˜8 hours with shaking at 30° C. Thus, paTF130system represents circumstances in which the transcription of both lightchain and heavy chain are simultaneously induced. On the other hand, thepxTF-7T3FL system was cultured under conditions designed to temporallyseparate the expression of each chain by first inducing the phoApromoter, controlling light chain transcription, and then at a latertime inducing the tacII promoter, controlling heavy chain transcription.

Non-reduced whole cell lysates from induced cultures were prepared asfollows: (1) 1 OD₆₀₀-ml pellets were centrifuged in a microfuge tube;(2) each pellet was resuspended in 90 ul TE (10 mM Tris pH 7.6. 1 mMEDTA); (3) 10 ul of 100 mM iodoacetic acid (Sigma I-2512) was added toeach sample to block any free cysteines and prevent disulfide shuffling;(4) 20 ul of 10% SDS was added to each sample. The samples werevortexed, heated to about 90° C. for ˜3 minutes and then vortexed again.After the samples had cooled to room temperature, ˜750 ul acetone wasadded to precipitate the protein. The samples were vortexed and left atroom temperature for about 15 minutes. Following centrifugation for 5minutes in a microcentrifuge, the supernatant of each sample wasaspirated off and each protein pellet was resuspended in 50 ul dH₂O+50ul 2× NOVEX sample buffer. The samples were then heated for ˜3-5 minutesat about 90° C., vortexed well and allowed to cool to room temperature.A final 5 minute centrifugation was then done and the supernatants weretransferred to clean tubes.

Reduced samples were prepared by following steps similar to what isdescribed above for non-reduced samples, except that 10 ul of 1M DTT wasadded to the cell resuspension solution in Step (2) and the addition ofIAA was omitted in Step (3). Reducing agent was also added to aconcentration of 100 mM when the protein precipitate was resuspended in2× sample buffer+dH₂O.

Following preparation, 5 ul of each sample was loaded onto a 10 well,1.0 mm NOVEX manufactured 12% Tris-Glycine SDS-PAG and electrophoresedat ˜120 volts for 1.5-2 hours. The resulting gels were used forimmunoblot analysis.

For immunoblot analysis, the SDS-PAGE gels were electroblotted ontonitrocellulose membranes (NOVEX). The membranes were then blocked usinga solution of 1× NET (150 mM NaCl, 5 mM EDTA, 50 mM Tris pH 7.4, 0.05%Triton X-100)+0.5% gelatin for approximately 30 min.-1 hour rocking atroom temperature. Following the blocking step, the membranes were placedin a solution of 1× NET+0.5% gelatin+anti-Fab antibody(peroxidase-conjugated goat IgG fraction to human IgG Fab; CAPPEL#55223) for non-reduced samples or 1× NET+0.5% gelatin+anti-Fabantibody+anti-Fc antibody (Jackson Immuno Research Labs #109-035-008)for reduced samples. The anti-Fab antibody dilution ranged from 1:50,000to 1:1,000,000 depending on the lot of antibody and the anti-Fc antibodywas diluted 1:1,000,000. The membranes were left in the antibodysolution overnight at room temperature with rocking. The next morning,the membranes were washed a minimum of 3×10 minutes in 1× NET+0.5%gelatin and then 1×15 minutes in TBS.(20 mM Tris pH 7.5, 500 mM NaCl).The protein bands bound by the antibody were visualized by usingAmersham Pharmacia Biotech ECL detection and exposing the membrane toX-Ray film.

Results

Plasmids for production of anti-tissue factor IgG1, paTF130 andpxTF-7T3FL, were constructed, transformed into strain 33D3 and inducedas previously described. Non-reduced and reduced whole cell lysatesamples were then prepared and analyzed by immunoblot. The results areshown in FIG. 11A and FIG. 11B. Using TIR's of 7 for light chain and 3for heavy chain, a simultaneous induction of the promoters controllingthese genes results in a secretory block as demonstrated by the reducedsample for paTF130 (FIG. 11A). The accumulation of both heavy and lightchain precursors is clearly evident in this lane. Very little maturelight chain and mature heavy chain are detected and the majority of theprotein accumulates as precursor. However, once the expression of heavyand light chain are temporally separated, as in pxTF-7T3FL, asignificant quantity of mature light chain accumulates (FIG. 11A).Although a small amount of light chain precursor is still detected inthis sample, this level does not appear to cause problems for eitherlight chain or heavy chain secretion. In addition, temporal expressionleads to the efficient secretion of mature heavy chain, to asignificantly greater level than that obtained with paTF130, with noevidence of precursor accumulation.

The correlation of efficient secretion with assembly of the full-lengthantibody is shown by the non-reduced samples (FIG. 11B). Full-lengthantibody is detected in both samples; however, the quantity variesdramatically. As the arrow indicates, only a faint full-length band isdetected in the paTF130 sample. This band becomes much more prominent inthe pxTF-7T3FL sample.

Example 3 Production of Anti-Tissue Factor F(ab′)₂

In this example, a single promoter plasmid, pCYC56, was used as acontrol. pCYC56 is structurally analogous to pS1130, with the exceptionthat the insert sequence encodes for the light and heavy chain fragmentof an anti-Tissue Factor antibody. A dual promoter plasmid, pxTF-7T3,was created similar to the dual promoter plasmid pxCD18-7T3 of Example1, and used to enable temporal separation of anti-Tissue Factor lightchain and heavy chain expression. The lacI sequence from the plasmidpMS421 was also incorporated onto pxTF-7T3 to create a new dual promoterpJVG31L. The addition of lacI obviates the need for co-expression withpMS421.

The host strain used in these fermentations was a derivative of E. coliW3110, designated 60H4. The complete genotype of 60H4 is: W3110ΔfhuAΔmanA phoAΔE15 Δ(argF-lac)169 deoC2 degP41(ΔpstI-Kan^(r))IN(rrnD-rrnE) 1 ilvG2096(Val^(r)) Δprc pre-suppressor. The 60H4 hostcells were transformed with either pCYC56, pJVG31L or the combination ofpxTF-7T3 and pMS421 and successful transformants were selected and grownin culture.

Fermentations were run under conditions similar to those for anti-CD18F(ab′)₂ as described in Example 1, with the principle exceptions thatthe run length varied between approximately 72 and 114 hours, and heavychain was induced using IPTG from approximately 4 to 12 hours followingthe attainment of a culture OD550 of 220.

The protein G assay used for anti-CD18 F(ab′)₂ was also used to analyzethe anti-Tissue Factor F(ab′)₂ products, with the exception that ananti-Tissue Factor F(ab′)₂ standard was used to generate the standardcurve.

A series of anti-Tissue Factor F(ab′)₂ fermentation runs were conductedusing the promoter systems described above. The yields of assembledanti-Tissue Factor F(ab′)₂ complex increased from 1 g/L with the singlepromoter system to 2.6±0.3 g/L (n=13) using the dual promoter systemwith pJVG3IL.

Therefore, the results show that by temporally separating the light andheavy chain synthesis, a significantly increased yield of assembledanti-Tissue Factor F(ab′)₂ was obtained.

Although the forgoing refers to particular embodiments, it will beunderstood that the present invention is not so limited. It will occurto those ordinary skilled in the art that various modifications may bemade to the disclosed embodiments without diverting from the overallconcept of the invention. All such modifications are, intended to bewithin the scope of the present invention.

1. A process for producing a functional antibody or fragment thereof ina host cell transformed with two separate translational unitsrespectively encoding the light and heavy chains of said antibody orfragment thereof, comprising the steps of: a) culturing the host cellunder suitable conditions so that the light chain and heavy chain areexpressed in a sequential fashion, thereby temporally separating theproduction of the light and heavy chains; and b) allowing the assemblyof the light and heavy chains to form the functional antibody orfragment thereof.
 2. The process of claim 1 wherein the host cell isprokaryotic, each translational unit further comprising a nucleotidesequence encoding for a prokaryotic secretion signal or variant thereofoperably linked to the N′-terminal of the light or heavy chain.
 3. Theprocess of claim 2, wherein the two separate translational units arecontrolled by different promoters.
 4. The process of claim 3, whereinthe two translational units are located on a single recombinant vector.5. The process of claim 2, wherein the secretion signal is selected fromthe group consisting of STII, OmpA, PhoE, LamB, MBP and PhoA.
 6. Theprocess of claim 5, wherein the secretion signal is STII.
 7. The processof claim 3, wherein the promoter for each translational unit is selectedfrom the group consisting of phoA, TacI, TacII, lpp, lac-lpp, lac, ara,trp, trc and T7 promoters.
 8. The process of claim 7, wherein onepromoter is the phoA promoter and the other promoter is the TacIIpromoter.
 9. The process of claim 1, wherein the antibody fragment isselected from the group consisting of Fab, Fab′, F(ab′)₂,F(ab′)₂-leucine zipper, Fv and dsFv.
 10. The process of claim 1, whereinthe antibody is specific to an antigen selected from the groupconsisting of VEGF, IgE, CD11, CD18 and tissue factor (TF).
 11. Theprocess of claim 10, wherein the antibody is an anti-CD18 antibody or ananti-TF antibody.
 12. The process of claim 1, wherein the antibody is achimeric antibody.
 13. The process of claim 1, wherein the antibody is ahumanized antibody.
 14. The process of claim 1, wherein the antibody isa human antibody.
 15. The process of claim 1, wherein the host cell is aprokaryotic cell from an E. coli strain.
 16. The process of claim 15,wherein the E. coli strain is genetically engineered to over-express atleast one chaperone protein selected from the group consisting of DsbA,DsbC, DsbG and FkpA.
 17. The process of claim 15, wherein the E. colistrain is deficient for endogenous protease activities.
 18. The processof claim 15, wherein the genotype of the E. coli strain contains Δprcpre-suppressor.
 19. The process of claim 1, wherein the heavy chain ofthe antibody is full length.
 20. The process of claim 19, wherein thelight chain is expressed first and the full length heavy chain issubsequently expressed.
 21. A system for sequential expression of alight chain and a heavy chain of an antibody or fragment thereof,comprising a host cell transformed with a recombinant vector, saidvector comprising two separate translational units respectively encodingthe light chain and heavy chain, each translational unit operably linkedto a different promoter, wherein under suitable conditions, theactivation of the two translational units are temporally separated,thereby allowing the sequential expression of the light chain and theheavy chain.
 22. The system of claim 21 wherein the host cell isprokaryotic, wherein each translational unit further comprises anucleotide sequence encoding for a prokaryotic secretion signal operablylinked to the 5′-end of the nucleic acid encoding the light or heavychain.
 23. The system of claim 22, wherein the secretion signal isselected from the group consisting of STII, OmpA, PhoE, LamB, MBP andPhoA.
 24. The system of claim 23, wherein the secretion signal is STII.25. The system of claim 24, wherein two STII variants are usedrespectively in the two translational units to provide a translationalstrength combination of about (7-light, 3-heavy).
 26. The system ofclaim 21, wherein each translational unit is operably linked to adifferent inducible promoter.
 27. The system of claim 26, wherein theinducible promoter is selected from the group consisting of phoA, TacI,TacII, lpp, lac-lpp, lac, ara, trp, trc and T7 promoters.
 28. The systemof claim 27, wherein one promoter is the phoA promoter and the otherpromoter is the TacII promoter.
 29. The system of claim 21, wherein theantibody fragment is selected from the group consisting of Fab, Fab′,F(ab′)₂, F(ab′)₂-leucine zipper, Fv and dsFv.
 30. The system of claim21, wherein the antibody is specific to an antigen selected from thegroup consisting of VEGF, IgE, CD11, CD18 and tissue factor (TF). 31.The system of claim 30, wherein the antibody is an anti-CD18 antibody oran anti-tissue factor antibody.
 32. The system of claim 21, wherein theantibody is a chimeric antibody.
 33. The system of claim 21, wherein theantibody is a humanized antibody.
 34. The system of claim 21, whereinthe antibody is a human antibody.
 35. The system of claim 21, whereinthe host cell is a prokaryotic cell from an E. coli strain.
 36. Thesystem of claim 35, wherein the E. coli strain is genetically engineeredto over-express at least one chaperone protein selected from the groupconsisting of DsbA, DsbC, DsbG and FkpA.
 37. The system of claim 35,wherein the E. coli strain is deficient for endogenous proteaseactivities.
 38. The system of claim 37, wherein the genotype of the E.coli strain contains Δprc pre-suppressor.
 39. The system of claim 21,wherein the heavy chain of the antibody is full length.
 40. The systemof claim 39, wherein the light chain is expressed first and the fulllength heavy chain is subsequently expressed.
 41. A recombinant vectorfor production of a functional antibody or fragment thereof in a hostcell, said vector comprising a) a first promoter preceding a firsttranslational unit encoding a secretion signal operably linked to alight chain and b) a second promoter preceding a second translationalunit encoding a secretion signal operably linked to a heavy chain, saidfirst and second promoters are inducible under different conditions. 42.The recombinant vector of claim 41, wherein the first and secondpromoters are selected from the group consisting of phoA, TacI, TacII,lpp, lac-lpp, lac, ara, trp, trc and T7 promoters.
 43. The recombinantvector of claim 42, wherein the first promoter is the phoA promoter andthe second promoter is the TacII promoter.
 44. The recombinant vector ofclaim 41, wherein the secretion signal is selected from the groupconsisting of STII, OmpA, PhoE, LamB, MBP and PhoA.
 45. The recombinantvector of claim 44, wherein the secretion signal is a STII.
 46. Therecombinant vector of claim 41, wherein the antibody fragment isselected from the group consisting of Fab, Fab′, F(ab′)₂, Fv and dsFv.47. The recombinant vector of claim 46, wherein the antibody fragment isfused to a dimerization domain.
 48. The recombinant vector of claim 41,wherein the antibody is specific to an antigen selected from the groupconsisting of VEGF, IgE, CD11, CD18 and tissue factor (TF).
 49. Therecombinant vector of claim 48, wherein the antibody is an anti-CD18antibody.
 50. The recombinant vector of claim 49, wherein the antibodyis an anti-CD18 F(ab′)₂-leucine zipper fusion.
 51. The recombinantvector of claim 50, said antibody having a light chain of SEQ ID NO:1.52. The recombinant vector of claim 50, said antibody having a heavychain of SEQ ID NO:2.
 53. The recombinant vector of claim 50, which is apxCD18-7T3 vector comprising the nucleic acid sequence of SEQ ID NO: 3.54. The recombinant vector of claim 48, wherein the antibody is ananti-TF antibody.
 55. The recombinant vector of claim 54, said antibodyhaving a light chain of SEQ ID NO:4.
 56. The recombinant vector of claim54, said antibody having a heavy chain of SEQ ID NO:5.
 57. Therecombinant vector of claim 54, which is a pxTF-7T3FL vector comprisingthe nucleic acid sequence of SEQ ID NO:6.